Methods and compositions for gene-specific demethylation by DNA methyltransferase (DNMT)-RNA interaction

ABSTRACT

The present invention relates to chimeric RNA oligonucleotides that are single-stranded oligonucleotides. These compounds are capable of targeting particular genes and reducing DNA methyltransferase activity. Accordingly, these compounds are particularly useful in the treatment of disease associated with aberrant DNA methyltransferase activity, such as cancer or a genetic disorder.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is the U.S. National Stage of InternationalApplication No. PCT/US2012/033617, filed Apr. 13, 2012, which claims thebenefit of U.S. Provisional Application No. 61/475,566, filed Apr. 14,2011, both of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

The invention relates to gene-specific chimeric RNA oligonucleotides andvarious uses thereof, including methods of treating cancer.

DNA methylation plays a significant role in mediating gene expression.Less is known about how this epigenetic mark is distributed throughoutthe genome, and in particular why DNA methyltransferases (DNMTs), whichare not sequence-specific, “avoid” certain CpG islands. Thedemethylating agent 5-aza-cytidine and analogs have been used forepigenetic research and received approval by the U.S. Food and DrugAdministration for the treatment of all subtypes of myelodysplasticsyndrome (MDS).

Currently, the two most prominent, approved demethylating agents for thetreatment of myelodysplastic syndrome are azacitidine (or 5-azacytidine,sold as Vidaza) and decitabine (or 5-aza-2′-deoxycytidine, sold asDacogen). These agents are incorporated into genomic DNA and inhibitDNMT by covalent binding. Cytotoxic effects have been associated withglobal incorporation of these agents into DNA and, thus, limit theirclinical application. Although non-nucleoside inhibitors of DNAmethylation possess less potential side effects, these inhibitors aregenerally less efficient than their nucleoside analogues and still causeglobal demethylation. Global, non-specific demethylation can lead toincreased tumorogenicity because demethylation likely decreasesexpression of tumor suppressor genes as well as prometastatic genes.

Therapy of diseases, including various cancers, can be impaired bynon-specific demethylating agents. Thus, new strategies forgene-specific delivery of agents to reduce methylation of DNA aredesired.

SUMMARY OF THE INVENTION

We have now developed chimeric RNA oligonucleotides (CROs) capable oftargeting specific genes and reducing DNA methylation of a gene by a DNAmethyltransferase. These CROs can be used to treat any condition havingaberrant hypermethylation, including cancer.

In a first aspect, the invention features a synthesized RNAoligonucleotide for reducing DNA methylation of a gene by a DNAmethyltransferase (DNMT), where the oligonucleotide includes a sequenceof about 15 to about 30 nucleotides (e.g., 15-20 nucleotides, 15-25nucleotides, 15-30 nucleotides, 15-35 nucleotides, 16-20 nucleotides,16-25 nucleotides, 16-30 nucleotides, 16-35 nucleotides, 17-20nucleotides, 17-25 nucleotides, 17-30 nucleotides, 17-35 nucleotides,18-20 nucleotides, 18-25 nucleotides, 18-30 nucleotides, or 18-35nucleotides) having at least 80% complementarity (e.g., at least being85%, 90%, 95%, 97%, 99%, and 100% complementarity) to a portion of anextra-coding RNA of the gene, and where the synthesized RNA binds to theextra-coding RNA to form a complex and the complex binds to a DNMT toreduce DNA methylation of the gene. In further embodiments, thesynthesized RNA oligonucleotide is covalently or non-covalently linkedto a targeting moiety, where the targeting moiety is a polypeptide,polypeptide derivative, or peptidomimetic that is capable of transportacross into a particular cell type (e.g., a cell-penetrating peptide,such as a polycationic or an amphipathic peptide).

In some embodiments, the invention features a synthesized RNAoligonucleotide for reducing DNA methylation of a gene by a DNAmethyltransferase (DNMT), where the oligonucleotide consists of asequence of about 15 to about 30 nucleotides (e.g., 15-20 nucleotides,15-25 nucleotides, 15-30 nucleotides, 15-35 nucleotides, 16-20nucleotides, 16-25 nucleotides, 16-30 nucleotides, 16-35 nucleotides,17-20 nucleotides, 17-25 nucleotides, 17-30 nucleotides, 17-35nucleotides, 18-20 nucleotides, 18-25 nucleotides, 18-30 nucleotides, or18-35 nucleotides) having at least 80% complementarity (e.g., at leastbeing 85%, 90%, 95%, 97%, 99%, and 100% complementarity) to a portion ofan extra-coding RNA of the gene, and where the synthesized RNA binds tothe extra-coding RNA to form a complex and the complex binds to a DNMTto reduce DNA methylation of the gene. In further embodiments, In someembodiments, the invention features a synthesized RNA oligonucleotidefor reducing DNA methylation of a gene by a DNA methyltransferase(DNMT), where the oligonucleotide consists of a sequence of about 15 toabout 30 nucleotides (e.g., 15-20 nucleotides, 15-25 nucleotides, 15-30nucleotides, 15-35 nucleotides, 16-20 nucleotides, 16-25 nucleotides,16-30 nucleotides, 16-35 nucleotides, 17-20 nucleotides, 17-25nucleotides, 17-30 nucleotides, 17-35 nucleotides, 18-20 nucleotides,18-25 nucleotides, 18-30 nucleotides, or 18-35 nucleotides) having atleast 80% complementarity (e.g., at least being 85%, 90%, 95%, 97%, 99%,and 100% complementarity) to an extra-coding RNA of the gene that iscovalently or non-covalently linked to a targeting moiety (e.g., asdescribed herein).

In a second aspect, the invention features a pharmaceutical compositionincluding any synthesized RNA oligonucleotide described herein and apharmaceutically acceptable excipient. In some embodiments of thisaspect, the composition further includes a histone deacetylase inhibitor(e.g., a hydroxamic acid, such as trichostatin A (TSA), vorinostat(SAHA), belinostat (PXD101),((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide(LAQ824), panobinostat (LBH589), suberoylanilide hydroxamic acid (SAHA),oxamflatin, scriptaid, suberic bishydroxamic acid (SBHA),m-carboxy-cinnamic acid bishydroxamic acid (CBHA), and pyroxamide); acyclic peptide, such as trapoxin A, apidicin, TPX-HA, and depsipeptide(FR901228)); a benzamide, such as entinostat (MS-275), N-acetyldinaline(CI994), and mocetinostat (MGCD0103); an electrophilic ketone, such astrifluoromethyl ketones or alpha-ketoamides, see Frey et al., Bioorg.Med. Chem. Lett. 12:3443-3447, 2002, and U.S. Pat. No. 6,511,990,incorporated herein by reference); and a fatty acid, such as valproicacid, arginine butyrate, butyric acid, and phenylbutyrate).

In a third aspect, the invention features a method of treating orprophylactically treating a subject having cancer, the method includingadministering to the subject any synthesized RNA oligonucleotidedescribed herein or any pharmaceutical composition described herein inan amount sufficient to treat the cancer. In some embodiments, thecancer is selected from the group of a myelodysplastic syndrome (e.g.,refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, refractory cytopenia with multilineagedysplasia, myelodysplastic syndrome associated with an isolated del(5q)chromosome abnormality, and a myeloproliferative neoplasm), leukemia(e.g., acute myeloid leukemia), head and neck cancer, liver cancer(e.g., hepatoma and hepatocellular carcinoma), lung cancer (e.g.,adenocarcinoma, small cell lung cancer, and non-small cell lung cancer),prostate cancer (e.g., adenocarcinoma), skin cancer (e.g., squamous cellcarcinoma), retinoblastoma, glioblastoma, breast cancer, thyroid cancer,ovarian cancer, pancreatic cancer, brain cancer, kidney cancer, coloncancer, endometrial cancer, gastric cancer, multiple myeloma, andlymphoma (e.g., T-cell lymphoma).

In a fourth aspect, the invention features a method of treating orprophylactically treating a subject having a genetic disorder, themethod including administering to the subject any synthesized RNAoligonucleotide described herein or any pharmaceutical compositiondescribed herein in an amount sufficient to treat the genetic disorder.In some embodiments, the genetic disorder is an imprinting disorder(e.g., Beckwith-Wiedemann Syndrome (BWS), Prader-Willi Syndrome (PWS),Angelman Syndrome (AS), Albright hereditary osteodystrophy (AHO),pseudohypoparathyroidism type 1A (PHP-IA), and pseudohypoparathyroidismtype 1B (PHP-IB)); a disorder associated with loss of imprinting (LOI)(e.g., LOI in IGF2/H19 for Wilms' tumor); and a repeat instabilitydisease (e.g., Fragile X syndrome and myotonic dystrophy).

In a fifth aspect, the invention features a method of preparing asynthesized RNA oligonucleotide for reducing DNA methylation of a geneby a DNA methyltransferase (DNMT), the method including preparing asequence of about 15 to about 30 nucleotides (e.g., 15-20 nucleotides,15-25 nucleotides, 15-30 nucleotides, 15-35 nucleotides, 16-20nucleotides, 16-25 nucleotides, 16-30 nucleotides, 16-35 nucleotides,17-20 nucleotides, 17-25 nucleotides, 17-30 nucleotides, 17-35nucleotides, 18-20 nucleotides, 18-25 nucleotides, 18-30 nucleotides, or18-35 nucleotides) having at least 80% complementarity (e.g., at leastbeing 85%, 90%, 95%, 97%, 99%, and 100% complementarity) to anextra-coding RNA of the gene. In a further embodiment of this aspect,the method further includes incorporating one or more modifiednucleotides into the sequence of about 15 to about 30 nucleotides.

In a sixth aspect, the invention features a method of identifying a RNAoligonucleotide for reducing DNA methylation of a gene by inactivating aDNA methyltransferase (DNMT), the method including: analyzing thesequence of one or more extra-coding RNAs of the gene; identifying anucleotide sequence having at least 80% complementarity (e.g., at leastbeing 85%, 90%, 95%, 97%, 99%, and 100% complementarity) to theextra-coding RNA; and determining one or more binding sites between thenucleotide sequence having at least 80% complementarity with theextra-coding RNA and/or the DNMT (e.g., by using a RNA electrophoreticmobility shift assay), thereby identifying the RNA oligonucleotide forreducing DNA methylation (e.g., by at least 10% or any percentagedescribed herein). In a further embodiment of this aspect, the methodincludes sequencing one or more extra-coding RNAs of the gene. Inanother further embodiment of this aspect, the method includessynthesizing the nucleotide sequence having at least 80%complementarity. In yet another further embodiment, the method includescontacting the nucleotide sequence having at least 80% complementaritywith one or more extra-coding RNAs and/or DNMTs. In another embodiment,the method includes the nucleotide sequence having one or more modifiednucleotides. In any of these embodiments, the method includes reducingDNA methylation of a gene by inactivating and sequestering a DNMT. Inanother embodiment, the CROs contain gene-specific sequences that can bedesigned having a sequence that is at least 80% (e.g., at least 85%,90%, 95%, 96%, 97%, 98%, 99% or more) complementary to a region spanningthe promoter, the coding part of the gene, and/or the 3′-downstream part(e.g., −2 kilobases and/or +2 kilobases from the transcriptional startsite (TSS) or the transcriptional end site (TES) of the genes,respectively). In a further embodiment of this aspect, the genes arethose contained in cluster C and any described herein.

In a seventh aspect, the invention features a method of diagnosing asubject for treatment with a synthesized RNA oligonucleotide forreducing DNA methylation of a gene by a DNA methyltransferase (DNMT),the method including: determining the subject as having a cancer or agenetic disorder (e.g., any cancer or disorder described herein) relatedto the gene (e.g., any gene described herein, such as from cluster C),analyzing the sequence of one or more extra-coding RNAs of the gene,identifying an RNA oligonucleotide sequence having at least 80%complementarity (e.g., at least being 85%, 90%, 95%, 97%, 99%, and 100%complementarity) to the extra-coding RNA, synthesizing the RNAoligonucleotide, and administering the RNA oligonucleotide for reducingthe methylation (e.g., by at least 10% or any percentage describedherein).

In particular embodiments of any of the above aspects, the extra-codingRNA is a transcribed RNA corresponding to a coding region, a non-codingregion, or both coding and non-coding regions, or is a fragment thereof.In particular embodiments, the extra-coding RNA is a transcribed RNAcorresponding to a non-coding region or a fragment of a non-codingregion. In other embodiments, the extra-coding RNA is a transcribed RNAcorresponding to both a coding region and a non-coding region or to botha fragment of a coding region and a fragment of a non-coding region.

In certain embodiments of any of the above aspects, the RNAoligonucleotide includes one or more modified nucleotides selected from5-azacytidine, 5-aza-5,6-dihydrocytosine, 5-aza-2′-deoxycytidine,beta-L-5-azacytidine, 2′-deoxy-beta-L-5-azacytidine,2′-deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine,5-fluorocytidine, 1-β-D-arabinofuranosil-5-azacytosine, and1-β-D-ribofuranosyl-2 (1H)-pyrimidinone, and analogs thereof (e.g.,analogs having one of the following substitutions for the hydrogen ofthe 4-amino group of the cytosine ring: methyl, ethyl,9-fluorenylmethyl, 9-(2-sulfo)fluorenylmethyl,9-(2,7-dibromo)fluorenylmethyl, 17-tetrabenzo[a,c,g,i]fluorenylmethyl,2-chloro-3-indenylmethyl, benz[f]inden-3-ylmethyl,2,7-di-tert-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)methyl,1,1-dioxobenzo[b]thiophene-2-ylmethyl, 2,2,2-trichloroethyl,2-trimethylsilylethyl, 2-phenylethyl, 1-(1-adamantyl)-1-methylethyl,2-chloroethyl, 1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromethyl,1,1-dimethyl-2,2,2-trichloroethyl, 1-methyl-1-(4-biphenylyl)ethyl,1-(3,5-di-tert-butylphenyl)-1-methylethyl, 2-(2′- and 4′-pyridyl)ethyl,2,2-bis(4′-nitrophenyl)ethyl, N-(2-pivaloylamino)-1,1-dimethylethyl,2-[(2-nitrophenyl)dithio]-1-phenylethyl,2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, 2-adamantyl,vinyl, allyl, 1-isopropylallyl, cinnamyl, 4-nitrocinnamyl,3-(3′-pyridyl)prop-2-enyl, 8-quinolyl, N-hydroxypiperidinyl,alkyldithio, benzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,p-chlorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl,9-anthrylmethyl, diphenylmethyl, 2-methylthioethyl,2-methylsulfonylethyl, 2-p-toluenesulfonyl)ethyl,[2-(1,3-dithianyl)]methyl, 4-methylthiophenyl, 2,4-dimethylthiophenyl,2-phosphonioethyl, 1-methyl-1-(triphenylphosphonio)ethyl,1,1-dimethyl-2-cyanoethyl, 2-dansylethyl, 4-phenylacetoxybenzyl,4-azidobenzyl, 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl,p-(dihydroxyboryl)benzyl, 5-benzisoxazolylmethyl,2-(trifluoroethyl)-6-chromonylmethyl, m-nitrophenyl,3,5-dimethoxybenzyl, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl,α-methylnitropiperonyl, o-nitrophenyl, 3,4-dimethoxy-6-nitrobenzyl,phenyl(o-nitrophenyl)ethyl, 2-(2-nitrophenyl)ethyl, 6-nitroveratryl,4-methoxyphenacyl, 3′,5′-dimethoxybenzoin, t-amyl, S-benzylthio,butynyl, p-cyanobenzyl, cyclohexyl, cyclopentyl, cyclopropylmethyl,p-decyloxybenzyl, diisopropylmethyl, 2,2-dimethoxycarbonylvinyl,o-(N,N-dimethylcarboxamido)benzyl,1,1-dimethyl-3-(N,N-dimethycarboxamido)propyl, 1,1-dimethylpropynyl,2-furanylmethyl, 2-iodoethyl, isobomyl, isobutyl, isonicotinyl,p-(p′-methoxyphenylazo)benzyl, 1-methylcyclobutyl, 1-methylcyclohexyl,1-methyl-1-cyclopropylmethyl, 1-methyl-1-(p-phenylazophenyl)ethyl,1-methyl-1-phenylethyl, 1-methyl-1-(4′-pyridyl)ethyl, phenyl,p-(phenylazo)benzyl, 2,4,6-tri-t-butylphenyl,4-(trimethylammonium)benzyl, 2,4,6-trimethylbenzyl, a urea (e.g., a ureawith phenothiazinyl-(10)-carbonyl, N′-p-toluenesulfonylaminocarbonyl,and N′-phenylaminothiocarbonyl), and an amide (e.g., formamide,acetamide, phenoxyacetamide, trichloroacetamide, trifluoroacetamide,phenyacetamide, 3-phenylpropamide, pent-4-enamide,o-nitrophenylacetamide, o-nitrophenoxyacetamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide,3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide,o-(benzoyloxymethyl)benzamide,2-[(t-butyldiphenylsiloxy)methyl)methyl]benzamide,3-(3′,6′-dioxo-2′,4′,5′-trimethylcyclohexa-1′,4′-diene)-3,3-dimethylpropionamide,o-hydroxy-trans-cinnamide, acetoacetamide, p-toluenesulfonamide, andbenzesulfonamide); or those having one of the following substitutionsfor the 4-amino group of the cytosine ring: 4-O-methoxy and4-S-methylsulfanyl). In further embodiments, the modified nucleotide isa cytidine analog further conjugated to a guanosine nucleotide (e.g.,5-aza-2′-deoxycytidine-phosphodiester linkage-guanosine,5-aza-2′-deoxycytidine-phosphodiester linkage-2′-deoxy-guanosine,guanosine-phosphodiester linkage-5-aza-2′-deoxycytidine, or2′-deoxy-guanosine-phosphodiester linkage-5-aza-2′-deoxycytidine). Inother embodiments, the RNA oligonucleotide includes one or more modifiednucleotides selected from cytarabine, fludarabine, gemcitabine,cladribine, clofarabine, 5-fluorouracil, azathioprine, floxuridine,mercaptopurine, thioguanine, pentostatin, and cladribine, or an analogthereof. In yet further embodiments, the modified nucleotide is5-azacytidine or 5-aza-2′-deoxycytidine.

In other embodiments of any of the above aspects, the RNAoligonucleotide includes a therapeutic agent selected from ademethylating agent (e.g., 5-azacytidine (azacitidine),5-aza-2′-deoxycytidine (decitabine), any cytidine analog describedherein, and any other demethylating agent described herein), a DNAand/or RNA polymerase inhibitor (e.g., cytarabine, fludarabine,gemcitabine, cladribine, and clofarabine), a thymidylate synthaseinhibitor (e.g., 5-fluorouracil, floxuridine, capecitabine, tegafur, andcarmofur), an immunosuppressant (e.g., azathioprine), an adenosinedeaminase inhibitor (e.g., pentostatin), a thiopurine (e.g., thioguanineand mercaptopurine), or a label (e.g., an isotope, such as a positronemitting isotope; a radioimaging agent or a radiolabel; a fluorescentlabel, such as green fluorescent protein (GFP), fluorescein, andrhodamine; a nuclear magnetic resonance active label; a luminescentlabel; a chromophore label; a chemiluminescence label, such asluciferase and β-galactosidase; an enzymatic label, such as peroxidaseand phosphatase; a reporter molecule, such as biotin or a histamine tag;and an antibody or an antibody fragment).

In other embodiments of any of the above aspects, the gene is C/EBPa(CCAAT enhancer binding protein alpha); SPE (spleen focus forming virus(SFFV) proviral integration oncogene spi1); RXRA (retinoid X receptor,alpha); RARB (retinoic acid receptor, beta); RB1 (retinoblastoma 1);CDKN2A (cyclin-dependent kinase inhibitor 2A); CDH1 (cadherin 1, type 1,E-cadherin); CDH13 (cadherin 13, H-cadherin); TIMP3 (TIMPmetallopeptidase inhibitor 3); VHL (von Hippel-Lindau tumor suppressor);MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2); MGMT(O-6-methylguanine-DNA methyltransferase); BRCA1 (breast cancer 1, earlyonset); GSTP1 (glutathione S-transferase pi 1); HLTF (helicase-liketranscription factor); RASSF1 (Ras association (RalGDS/AF-6) domainfamily member 1); SOCS1 (suppressor of cytokine signaling 1); ESR1(estrogen receptor 1); or DAPK1 (death-associated protein kinase 1). Insome embodiments, the gene is C/EBPa and the extra-coding RNA of C/EBPais SEQ ID NO:1 or SEQ ID NO:8, or a fragment thereof. In otherembodiments, the gene is SPI1 and the extra-coding RNA of SPI1 is SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or a fragment thereof.In yet other embodiments, the gene is RXRA and the extra-coding RNA ofRXRA is SEQ ID NO:6 or SEQ ID NO:7, or a fragment thereof. In someembodiments, the gene is RARB and the extra-coding RNA of RARB is SEQ IDNO:9, or a fragment thereof. In other embodiments, the extra-coding RNAof the gene is the promoter region of the gene. In further embodiments,the extra-coding RNA is the promoter region of the gene selected fromC/EBPa, SPI1, RXRA, and RARB.

In other embodiments of any of the above aspects, the genes are found incluster C as described herein. Some of those genes belong to particularGene Ontology (GO) categories (www.geneontology.org and The GeneOntology Consortium, “Gene ontology: tool for the unification ofbiology,” Nat. Genet. 25(1):25-29 (2009)). Exemplary non-limiting GOcategories include genes related to conversion of one or more primaryRNA transcripts into one or more mature RNA molecules (GO:0006396, RNAprocessing); genes related to biochemical and morphological phases thatoccur during successive cell or nuclear replication events (GO:0007049,cell cycle), genes related to chemical reactions and pathways involvingmRNA (GO:0016071, mRNA metabolic process), genes related to assembly,arrangement, or disassembly of chromosomes (GO:0051276, chromosomeorganization), genes related to splicing and joining primary RNAtranscript to form mature form of the RNA (GO:0008380, RNA splicing),and any described herein.

In particular embodiments of any of the above aspects, theoligonucleotide is not double-stranded.

In some embodiments of any of the above aspects, the DNMT is selectedfrom the group consisting of DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L.In further embodiments, the DNMT is DNMT1.

DEFINITIONS

By “chimeric RNA oligonucleotide” or “CRO” is meant an RNAoligonucleotide capable of reducing the activity of DNAmethyltransferase and of selectively or specifically hybridizing to atarget sequence within a gene locus.

By “complementarity” is meant an oligonucleotide able to form one ormore hydrogen bonds with another oligonucleotide (e.g., a RNA) by eithertraditional Watson-Crick (e.g. G with C, A with T, or A with U) or othernon-traditional types (e.g., diaminopurine with T, 5-methyl C with G,2-thiothymidine with A, inosine with C, pseudoisocytosine with G, etc.).In reference to the oligonucleotide of the present invention, thebinding site (or binding energy) for a nucleotide with its target orcomplementary sequence is sufficient to allow the relevant function ofthe nucleotide to proceed, e.g., targeting of the RNA, binding to DNMT,or triple helix formation. Determination of a binding site (or bindingenergy) for a nucleotide is well known in the art (see, e.g., Turner etal., Cold Spring Harbor Symp. Quant. Biol. 52:123-133, 1987; Frier etal., Proc. Natl. Acad. Sci. USA 83:9373-9377, 1986; and Turner et al.,J. Am. Chem. Soc. 109:3783-3785, 1987). A percent complementarityindicates the percentage of contiguous residues in a nucleotide that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleotide (e.g., being 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99%, and 100% complementary), optionally determined understringent conditions.

By “complex binds to a DNA methyltransferase” is meant an interactionbetween a CRO-extra-coding RNA complex and a DNA methyltransferase witha dissociation constant (Kd) measured in the range of between 0.01 μM to0.10 μM (e.g., 0.01, 0.02, 0.03, 0.05, 0.08, 0.09, and 0.1 μM).

By “cytidine analog” is meant a modified nucleotide having a cytosinebase or a modified cytosine base.

By “demethylating agent” is meant an agent that leads to decreasedmethylation of DNA by directly or indirectly inactivating a DNAmethyltransferase.

By “DNA methyltransferase” or “DNMT” is meant an enzyme that methylatesthe C-5 carbon of cytosines in DNA. Exemplary DNMTs include DNMT1,DNMT2, DNMT3a, DNMT3b, and DNMT3L.

By “extra-coding RNA” or “ecRNA” is meant transcribed pre-mRNA includingboth coding and non-coding regions or only non-coding regions.Extra-coding RNA regions and fragments thereof may include those thatcan form secondary structures with CROs as predicted by RNA secondarystructure prediction programs known in the art (e.g., CentroidFold,Mfold, RNAfold, RNAstructure, and CONTRAfold). Extra-coding RNA regionsand fragments thereof may also include those that modulate methylationpatterns of genes associated with the extra-coding RNA region. In someembodiments, the ecRNA only includes one or more contiguous, non-codingregions of a gene.

By “fragment” is meant a portion of a full-length amino acid or nucleicacid sequence (e.g., any sequence described herein). Fragments mayinclude at least 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, 18, 20, 25, 30,35, 40, 45, or 50 contiguous amino acids or nucleic acids of the fulllength sequence. A fragment may retain at least one of the biologicalactivities of the full length protein or nucleic acid.

By “inactivating a DNA methyltransferase” is meant a RNA oligonucleotidethat reduces the methylating activity of DNA methyltransferase by atleast 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99%) compared to acontrol lacking the RNA oligonucleotide.

By “modified nucleotide” is meant a nucleotide having a modification tothe chemical structure of one or more of the base, sugar, and backbone,including the phosphate linker.

By “reduced DNA methylation” is meant reducing the methylating activityof DNA methyltransferase by at least 10% (e.g., at least 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, oreven 99%), compared to a control lacking the RNA oligonucleotide.Non-limiting methods for determining reduced DNA methylation aredescribed herein (e.g., in Example 4).

By “stringent conditions” is meant conditions under which a RNAoligonucleotide will selectively or specifically hybridize to its targetsequence (i.e., an extra-coding RNA), typically in a complex mixture ofnucleic acids, but to no other sequences. Stringent conditions aresequence-dependent and length-dependent. Generally, stringent conditionsare selected to be about 5° C. to about 25° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. Stringent conditions may also include destabilizing agents,such as formamide. For selective or specific hybridization, a positivesignal is at least two times background, preferably 10 times backgroundhybridization. Exemplary stringent conditions include: 50% formamide,4×SSC, and 1% SDS, incubating at 42° C.; and 4×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Hybridizationtechniques are generally described in Nucleic Acid Hybridization, APractical Approach (eds. B. D. Hames and S. J. Higgins, IRL Press,1985); Tijssen, “Overview of principles of hybridization and thestrategy of nucleic acid assays” in Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization with Nucleic Probes(ed. P. C. van der Vliet, Elsevier Science Publishers B.V., 1993); PCRProtocols, A Guide to Methods and Applications (eds. M. A. Innis et al.,Academic Press, Inc., New York, 1990); Gall and Pardue, Proc. Natl.Acad. Sci., USA 63:378-383, 1969; and John et al., Nature 223:582-587,1969.

By “substantially identical” is meant an oligonucleotide exhibiting atleast 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or even 99% identity to a reference nucleotide sequence. Forpolypeptides, the length of comparison sequences will generally be atleast 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 50, or 100) amino acids. For oligonucleotides, thelength of comparison sequences will generally be at least 5 nucleotides(e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). It is to beunderstood herein that gaps may be found between the nucleotide ofsequences that are identical or similar to nucleotides of the originaloligonucleotide. The gaps may include no nucleotides or one or morenucleotides that are not identical or similar to the originaloligonucleotide. Percent identity may be determined, for example, withan algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, using default gap weights.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “treating” a disease, disorder, or condition in a subject is meantreducing at least one symptom of the disease, disorder, or condition byadministrating a conjugate or therapeutic polypeptide to the subject.

By “prophylactically treating” a disease, disorder, or condition in asubject is meant reducing the frequency of occurrence or severity of(e.g., preventing) a disease, disorder or condition by administering tothe subject a conjugate or therapeutic polypeptide to the subject priorto the appearance of a disease symptom or symptoms.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1L show the characterization of an extra-coding RNA of theC/EBPa gene. FIG. 1A is a diagram of coding (gray arrow) andextra-coding (arrow labeled with “˜5 kb”) RNAs. The white rectangleindicates the position of the probe used in the RNA electrophoreticmobility shift assays (REMSAs). The black star indicates the C/EBPa mRNAprobe located in the 3′UTR region, and the white star indicates theextra-coding probe located after the polyA signal outside the codingsequence. FIG. 1B is a Northern blot hybridization analysis of codingRNAs (black star), extra-coding RNAs (white star), and loading control(EtBr) using the mRNA probe and extra-coding probe in HL-60, U937,Jurkat, and K562 cell lines. FIG. 1C show graphs for levels of coding(left) and extra-coding (right) C/EBPa transcripts in cellular fractionsfrom HL-60 cell lines. FIGS. 1D-1E show relative levels of thetranscripts in total and nuclear fractions, where the ratio between mRNAand ecRNA levels were 30:1 and 2:1 in total and nuclear fractions,respectively. FIG. 1F shows that DRB treatment did not reduce ecRNAlevels upon release of cells from double thymidine block. FIG. 1G showsthat treatment with the Pol III inhibitor ML-60218 significantly reducedlevels of ecRNA and Pol III-transcribed 5S rRNA and only moderatelyaffected C/EBPa mRNA (P=n.s.). FIGS. 1C-1G show results of qRT PCRassays. Statistical analysis performed by paired T-test (*P<0.05;**P<001; ***P<0.001); and all error bars are means±s.d (n=3). FIG. 1H isa series of Northern blots of non-coding (nc) C/EBPa transcriptsperformed on nuclear RNA fractions of three different cell lines (leftpanel). The two middle panels demonstrate enrichment of non-codingC/EBPa transcripts in nuclear polyA(−) fraction; the right panelillustrates the extent of polyA(−) fraction purity. FIG. 1I are resultsfrom primer extension experiments performed on total cellular andcellular polyA(−) RNAs of HL-60 cell line as described in theexperimental procedures. The AL16 oligonucleotide is located in thecoding region of C/EBPa gene. Primer extension revealed a ˜4 kb band inthe polyA(−) fraction, marking the TSS for non-coding transcript at ˜2kb upstream to the mRNA TSS. FIG. 1J are sequences used for mappingnon-coding C/EBPa transcripts by 5′ (SEQ ID NO:58), 3′ (SEQ ID NO:59)RACE in two cell lines, HL-60 and U937. R4, R6, R8, and AL21, A123, AL25are primers used in RACE. Also provided are the 5′ and 3′ ends ofnon-coding and coding transcripts in both cell lines. FIG. 1K showresults from double thymidine block of HL-60 cells. Treatment withthymidine (2.5 mM) arrested the cells in G1/S phase border. FACSanalysis shows synchronization after the treatment. FIG. 1L shows thelevels of coding and ecRNA upon release from double thymidine block.Induction of ecRNA preceded and surpassed expression of C/EBPa mRNA.

FIGS. 2A-2C show that down-regulation of C/EBPa extra-coding RNAs leadsto down-regulation of C/EBPa mRNA expression and increased methylationof the C/EBPa promoter region. FIG. 2A is a graph showing the effect ofshRNAs that target extra-coding RNAs on the levels of extra-coding RNA(left) and of mRNA expression (right). The shRNAs are designed againstthe sequence after the polyA signal. FIG. 2B is a chart showing theeffect of shRNAs that target extra-coding RNAs on promoter methylation.Methylation data from bisulfate sequencing was analyzed with BIQAnalyzer software (Aggregated Representation of Methylation Data). Thewhite bars represent methylated states, the black bars representunmethylated states of each CpG dinucleotide within the sequencingreads, and changes from black bars (as shown in lane labeled “Scrambled”in FIG. 2B) to white bars (as shown in lanes labeled “sh1,” “sh2,” and“sh3” in FIG. 2B) indicate increase in methylation states ofcorresponding CpG dinucleotides. FIG. 2C is a graph showingquantification of the results shown in FIG. 2B.

FIG. 3 shows immunoprecipitation of C/EBPa extra-coding RNAs withantibodies against DNA methyltransferase 1 (DNMT1) and with control(IgG). The upper panel shows the end-point of RT-PCR, and the bottompanel shows graphs having quantitated results from real time qRT-PCR.

FIGS. 4A-4F show that DNMT1 binds to folded RNAs with high affinitycomparable to that of DNA. FIG. 4A provides an exemplary RNAoligonucleotide (SEQ ID NO:60) corresponding to the 5′ end of C/EBPaextra-coding RNA. FIG. 4B provides stem-loop-like folding of the RNAoligonucleotide (SEQ ID NO:60). FIG. 4C provides an exemplarydouble-stranded DNA oligonucleotide corresponding to the 5′ sequence ofthe RNA oligonucleotide (SEQ ID NO:61). FIG. 4D shows an analysis of aRNA electrophoretic mobility shift assay (REMSA) for RNA and DNA.Provided are data for free probes (lanes 1 and 5); probes with DNMT1,without polydldC (lanes 2 and 6); and probes with DNMT1 havingincreasing amounts of competitor, polydldC (lanes 3, 4, 7, and 8). Thebottom panel demonstrates uniformity in probe loading. FIG. 4E showsthat double-stranded RNA (dsRNA) molecules bind to DNMT1 andsingle-stranded RNA (ssRNA) of the same primary structure have low tozero capacity for DNMT1. FIG. 4F shows an analysis of a REMSA with RNAseT3 digested transcripts corresponding to the 5′ regions of the C/EBPaextra-coding RNAs and the luciferase gene, showing similar bindingaffinity of DNMT1 to dsRNAs from different sources.

FIGS. 5A-5B show that transcription interferes with DNA methylationusing an in vitro assay. FIG. 5A are schematics showing (i) ahemimethylated DNA segment with an integrated 17 promoter; (ii) an invitro transcription assay using T7 polymerase and nucleotidetriphosphates (NTPs); (iii) a parallel in vitro transcription andmethylation assay using T7 polymerase, NTPs, and DNMT1; and (iv) an invitro methylation assay using NTPs and DNMT1. FIG. 5B shows a combinedbisulfite restriction analysis (COBRA) assay of methylation patternsacquired using the assays provided in FIG. 5A(ii)-(iv). The black arrowindicates the presence of digestion products and shows that DNMT1activity is present in the absence of transcription.

FIGS. 6A-6D show exemplary gene-specific RNA oligonucleotides forreducing DNA methylation. FIG. 6A is a schematic showing high-expressingRNAs that sequesters DNMT1 and interferes with DNA methylation of thecorresponding region (left). In absence of transcription (right), DNMT1methylates the targeted region. FIG. 6B is a schematic showing aproposed, non-limiting mechanism of DNMT1 sequestration by adouble-stranded chimeric RNA oligonucleotide. FIG. 6C is a schematicshowing a proposed non-limiting mechanism of DNMT1 sequestration throughforming a double-stranded complex between natural RNA and asingle-stranded chimeric RNA oligonucleotide. FIG. 6D depicts aproposed, non-limiting mechanism of DNMT1 sequestration through triplexstructure formation (e.g., H form) between genomic DNA and asingle-stranded chimeric RNA oligonucleotide. Aza indicates5-aza-2′-deoxycytidine or an analog thereof.

FIGS. 7A-7D show results from an exemplary screen for extra-coding RNAhaving “target site specificity.” FIG. 7A depicts the position of twochosen regions within C/EBPa extra-coding RNA: R1 and R2. BLAST analysesare provided for R2 (FIG. 7B) and R1 (FIG. 7C). FIG. 7D is a Northernblot hybridization analysis of probes corresponding to regions R1 andR2, where total RNAs was extracted from cell lines that express C/EBPa(HL-60 and U937 cell lines) and from cell lines that do not expressC/EBPa (HEK 293 and K562 cell lines).

FIG. 8 provides an exemplary sequence of the 3′-end of the C/EBPaextra-coding RNA (SEQ ID NO:1).

FIG. 9 provides exemplary extra-coding RNAs of four different locationsof the spleen focus forming virus proviral integration oncogene spiI(SPI1, Homo sapiens) gene locus, including bases 47356552-47356358 (SEQID NO:2), 47340202-47339789 (SEQ ID NO:3), 47342950-47342689 (SEQ IDNO:4), and 47340198-47339789 (SEQ ID NO:5) of Homo sapiens chromosome11, GRCh37.p2 primary reference assembly (NCBI Ref. Seq. NT_009237.18).

FIG. 10 provides exemplary extra-coding RNAs of two different locationsof the retinoid X receptor alpha (RXRA, Homo sapiens) gene locus,including bases 126630-127130 (SEQ ID NO:6) and 127305-127805 (SEQ IDNO:7) of Homo sapiens chromosome 9, GRCh37.p2 primary reference assembly(NCBI Ref. Seq. NT_019501.13).

FIGS. 11A and 11B provides an exemplary sequence of C/EBPa extra-codingRNA (SEQ ID NO:8).

FIG. 12 provides an exemplary sequence of retinoic acid receptor beta(RARB, Homo sapiens) extra-coding RNA (SEQ ID NO:9), including bases25407956-25410445 of Homo sapiens chromosome 3, GRCh37.p2 primaryreference assembly (NCBI Ref. Seq. NT_022517.18).

FIGS. 13A-13P show the loss- and gain-of-function studies demonstratingthat ecRNA maintains C/EBPa expression by regulating methylation of theC/EBPa locus. FIG. 13A is a diagram indicating the position of targetsequences for shRNA constructs (sh1-3; vertical arrows); the 1057 bpfragment derived from the ecRNA employed for overexpression (R1;double-headed arrow); and regions analyzed for changes in DNAmethylation (distal promoter; coding sequence, CDS; and 3′UTR; whitedouble-headed arrows). FIGS. 13B-13D show results of ecRNAloss-of-function, in which C/EBPa is expressed. FIG. 13B shows theeffect of ecRNA-targeting shRNAs on C/EBPa transcript levels in U937cells. FIG. 13C shows the effect of ecRNA-targeting shRNAs onmethylation of the C/EBPa promoter using bisulfite sequencing. Thelollipop representation is used to show methylation patterns, whereblack dots indicate methylated positions and white dots indicateunmethylated positions. FIG. 13D shows DNA methylation changes as theratios of methylated to unmethylated CpGs in all clones analyzed pereach construct. FIGS. 13E-13K show the results of ecRNA gain-of-functionstudies in K562 cells, in which C/EBPa is methylated and silenced. FIG.13E shows the effect of ecRNA upregulation on levels of C/EBPa mRNA. Theunrelated region (UR) is a 705 bp fragment located ˜45 kb downstream tothe C/EBPa gene. FIG. 13F shows the effect of ecRNA upregulation onmethylation of the C/EBPa locus, where lollipop representations (left)and DNA methylation changes (right) are provided. FIGS. 13G-13I showcomparisons of DNA methylation changes imposed by ecRNA overexpressionand 5-Aza-CR treatment by MassARRAY analysis. FIG. 13G is a diagramshowing the position of MassARRAY target regions; C/EBPa and C/EBPggenes; and CpG islands. FIG. 13H is a heatmap representing themethylation level of individual CpGs for overexpression samples (UR,R1); two untreated control cell lines (K562, HL-60); and mock5-Aza-CR-treated cells; below are snapshots of regions corresponding toC/EBPa and C/EBPg genes. FIG. 13I shows methylation changes (MethylationDelta) induced by ectopic ecRNA expression that are significant onlywithin C/EBPa locus. The region +6 kb includes the R1 fragment and wasexcluded from the statistical analysis. FIGS. 13J-13K are comparativeanalyses of C/EBPa and C/EBPg expression and methylation changesfollowing 5-Aza-CR treatment and ecRNA overexpression in K562 cells,where lollipop representations (left), DNA methylation changes (middle),and changes in expression levels determined by qRT PCR (right) areprovided. FIG. 13L is a diagram indicating the position of regions ofthe ecRNA employed for overexpression (R2 and R1) and unrelated regions(UR), and regions of C/EBPa gene analyzed for changes in DNA methylation(CDs and 3′UTR). FIG. 13M is a Northern blot analysis of untransfectedK562 and K562 stably transfected with constructs R1, UR, and EV (emptyvector) demonstrating uniformity of overexpression levels of R1 and URconstructs. FIG. 13N shows the effect of ecRNA upregulation onmethylation of the C/EBPa locus by COBRA analysis of the coding and 3′UTR regions of C/EBPa gene. Black arrows indicate incompletely digestedPCR products of bisulfite converted genomic DNAs isolated from cellsstably transfected with R1 and UR constructs. FIG. 13O shows the effectof ecRNA upregulation on levels of C/EBPa mRNA, where overexpression ofR1 led to significant upregulation of ecRNA. FIG. 13P shows comparativeanalyses of effects of ecRNA upregulation and 5-Aza-CR treatment on TP73promoter methylation and TP73 mRNA expression level, where lollipoprepresentations (left) and DNA methylation changes (right) are provided.For FIGS. 13H-13K and 13P, error bars indicate means±s.d.; all bisulfitesequenced clones were analyzed by Fisher's exact test; MassARRAY datawere analyzed by paired T-test; and *P<0.05; **P<0.001; ***P<0.001.

FIGS. 14A-14C are results from experiments showing that transcriptionimpedes DNA methylation. FIGS. 14A and 14B show lollipop representationsof bisulfite sequenced clones analyzed by Fisher's exact test (*P<0.05;**P<0.01; ***P<0.001). The right panels show DNA methylation changes asdescribed in FIG. 13D. The same effect was observed with two differentRNA polymerases: T7 and Sigma-Saturated (σ70)-Holoenzyme (E. coli RNApolymerase). FIG. 14C is a schematic showing the generation ofhemimethylated DNA. (a and b) The region corresponding to the 5′ regionof the ecRNA genomic template was amplified using a forward primercontaining the T7 promoter sequence and a biotinylated (arrowhead B) andnot-biotinylated reverse primers containing a unique BstX1 restrictionsite (unmethylated DNA; umDNA). (c) Biotinylated umDNA was in vitromethylated with M.SssI (NEB). (d) Efficiency of methylation was assessedby restriction digestion of the methylated DNA (mDNA) with methylationinsensitive and sensitive restriction enzymes MspI and HpaII,respectively. (e) 10× molar excess of not biotinylated umDNA was mixedwith biotinylated mDNA. The mixture was denatured (100° C.; 5 minutes),quickly chilled to 70° C., and reannealed by slow chilling down to 4° C.(0 The biotinylated DNAs, a ˜10:1 mixture of hmDNAs and mDNAs, werecaptured with streptavidin magnetic beads. (g) The beads were removedfollowing BstX1 restriction digestion. (h) COBRA analysis was performedto assess the purity of the hmDNA. The primers for bisulfite convertedDNA were designed against the upper (unmethylated) strand, hence BstU1Idigestion of hmDNA and umDNA should present identical restrictionpatterns. Presence of trace amount of BStUI digestion fragments in hmDNAlane (marked with asterisk) reflects ˜<10% of the mDNA in the mixture (eand f).

FIGS. 15A-15M show that DNMT1 binds to RNA with a greater affinity thanto DNA. FIG. 15A is a result from an RNA immunoprecipitation (RIP)demonstrating that ecRNA is immunoprecipitated with anti-DNMT1 antibodyin HL-60 cells. Error bars are means±s.d. FIG. 15B is a diagram showingthe position and sequences of RNA (SEQ ID NOs:62, 63) and dsDNAoligonucleotides used in EMSA. Asterisks indicate position of methylatedcytosines. umDNA (SEQ ID NOs:64, 71), hmDNA (SEQ ID NOs:65, 72), andmDNA (SEQ ID NOs:66, 73) refer to unmethylated, hemimethylated, andmethylated DNA probes, respectively. FIG. 15C are RNA and DNA EMSAperformed with a fixed concentration of ssRNA and dsDNAs (1 nM) andincreasing concentrations of DNMT1 protein as indicated above the gel.FIG. 15D is a nonlinear regression analysis of bound RNNDNA vs. DNMT1concentrations. Error bars indicate s.d. from two independent EMSAs.FIG. 15E shows the secondary structures predicted by RNAfold of ssRNAR05 (SEQ ID NO:62) and R04 (SEQ ID NO:63). FIG. 15F is a REMSA showingthat R04 displays lower DNMT1 affinity compared to R05 (FIG. 15C, leftpanel) at the same DNMT1 concentrations. FIG. 15G is a graph showingthat ecRNA specifically immunoprecipitates with anti-hDNMT1 antibody inU937 cells. FIG. 15H shows the enrichment of non-polyadenylated ecRNA inDNMT1-RNA precipitate (PolyA(−)) in HL-60 cells. The schematics outlinethe tested procedure and the TaqMan probe set (black double headedarrow) used to detect both C/EBPa transcripts (polyadenylated CEBPa mRNAand non-polyadenylated C/EBPa ecRNA). FIG. 15I is a diagram showingpositions and sequences of RNA (SEQ ID NO:60) and DNA (SEQ ID NOs:61,67, 70, 74) oligonucleotides used in EMSA, where possible stem-loop-likefolding within the ecRNA sequence is also shown. dsRNA oligo representsimperfect duplex between RNA oligo R01 and R03; double-stranded DNAoligo correspond to the sequences of the RNA oligo. FIG. 15J shows RNAand DNA EMSA performed with increasing amounts of competitors: polydl-dCand dsDNA. FIG. 15K shows secondary structures of R01 (SEQ ID NO:68) andmutated R01 (SEQ ID NO:69). Both are able to form stem-and-loop-likestructures. Asterisks indicate C to U substitutions. FIG. 15L is a gelshowing that DNMT1 binding to folded ssRNA is not affected by theabsence of CpG dinucleotides when stem-and-loop-like structure ispreserved. FIG. 15M is an RNA EMSA performed in the presence ofincreasing concentration of spermine, where no significant changes inbinding were observed.

FIGS. 16A-16H show the DNMT1 epitranscriptome analysis. FIG. 16A is apie chart showing distribution of specific DNMT1 library peaks. FIG. 16Bis a comparison of gene expression and methylation levels between DNMT1unbound and DNMT1 bound groups (P<0.0001). FIG. 16C shows a DNMT1Epitranscriptome map, where the cloud plots representing genes withinDNMT1 unbound and bound groups were stratified by methylation andexpression levels. FIG. 16D shows examples of genes falling into the C(C/EBPa) and B (USP29) clusters. Peaks are visualized using SISSRs (SiteIdentification from Short Sequence Reads) (Jothi et al., Nucleic AcidRes 36:5221-5231, 2008). FIG. 16E is a chart showing enrichment ofC/EBPa ecRNA in a cDNA library made of RNAs that co-immunoprecipitatedwith anti-hDNMT1 antibody. FIG. 16F is a flow chart of RIPseq analysisshowing various steps applied for the comparative RIPseq and RRBSanalyses. FIGS. 16G and 16H are additional examples of genes in clustersC and B.

FIG. 17 is a non-limiting model of putative long-distance DNMT1sequestration by ecRNA. Without being limited by theory, this modelproposes that ecRNA is transcribed from remote genomic template (locatednearby to coding genes 1 and 2) and maintain close physical proximitywith coding genes 3 and 4 by chromatin looping. This ecRNA, beingidentified by RIPseq, will likely be aligned to the respective “Codinggenes 1 and 2 loci” and not to the remote coding genes 3- and4-corresponding loci. The coding gene 5 belongs to cluster E, where itsexpression may be less likely to be affected by methylation (as detectedby RRBS).

FIG. 18 is a gene ontology (GO) chart of exemplary genes within clusterC. Enrichment is shown for the first 20 GO's biological processannotations, where enrichment was determined by using Benjaminicorrected P-values and −log₂ (P-value) are provided.

DETAILED DESCRIPTION

Contrary to traditional views that DNA methylation affects geneexpression, we have discovered that gene expression, i.e.,transcription, affects DNA methylation. Our discovery is based on theobservation that (i) double-stranded RNA molecules bind to DNAmethyltransferase (DNMT1), (ii) single-stranded RNA of the same primarystructure have low to zero capacity to bind DNMT1, and (iii) DNMT1 isinactive during transcription. Based on these observations, we developedsingle-stranded chimeric RNA oligonucleotides (CROs) having the dualability to inactivate DNA methyltransferase (DNMT) by enhanced bindingbetween an RNA sequence and DNMT and to target a specific gene duringtranscription by binding an extra-coding RNA or by triplex formationwith genomic DNA. Though the extra-coding RNA can include both codingand non-coding regions, we have discovered that non-coding regions of anextra-coding RNA may play a functional role in gene expression.

Without wishing to be limited by theory, we hypothesize that: (i)cell-type specific transcription is an underlying cause of the diversemethylation patterns; (ii) RNAs as physical entities could establish andmaintain cell-type specific methylation patterns; (iii) modulation ofexpression levels of selected transcripts may change methylationpatterns of respective genes and ultimately gene expression; and (iv)the enhanced ability of RNA to bind DNMT1 could be exploited in thedevelopment of a novel, therapeutic gene-specific demethylating agent.

Gene-specific CROs can be obtained by including unique genomic sequenceswith low capacity to form secondary structures. When the CRO form adouble-stranded duplex with their target, i.e., a naturally occurringRNA, the formed duplex will bind DNMT1 through its observed bindingcapacity to dsRNA. When the gene target is not available, such as whenthe RNA is not being transcribed, then the lack of secondary structurewill prevent CROs from non-specifically binding DNMT1. In this manner,the CROs target specific genes and avoid the global demethylating effectobserved with traditional demethylating agents. Non-limiting examples ofthe CRO interacting with transcribed RNA is provided in FIGS. 6C and 6D.

Furthermore, these chimeric RNA oligonucleotides can be used to deliveradditional demethylating agents. For example, 5-azacytidine and or5-aza-2′-deoxycytidine covalently bind DNMT1, and these known agents canbe incorporated into CROs to further enhance binding between DNMT andRNA sequences. For example, when the CRO includes 5-aza-2′-deoxycytidineand forms a double-stranded duplex with its gene target, then DNMT1 canbind via the secondary structure of the duplex and the5-aza-modification in cytidine.

Taken together, these chimeric RNA oligonucleotides can be used to treatvarious diseases associated with aberrant DNMT activity, such as cancer,and to develop a new field of customized, gene-targeted demethylatingtherapy.

Chimeric RNA Oligonucleotides

The chimeric RNA oligonucleotide (CRO) of the invention is capable ofreducing the activity of DNA methyltransferase and selectively orspecifically hybridizing to a target sequence in a gene and/or to acorresponding extra-coding RNA. The CRO includes a sequence of about 15to about 30 nucleotides having percentage complementarity to anextra-coding RNA of a gene. The formed duplex (with an extra-coding RNA)and/or triplex (with the respective genomic sequence) will allow forbinding to DNMT. This binding will result in sequence-specificprotection of the respective genomic sequences from the enzymaticactivity of DNMT. Accordingly, these chimeric RNA oligonucleotides canbe used for treatment (e.g., as an agent for cancer therapy) or fordiagnostic methods or kits (e.g., as a probe for a diagnostic assay).

The RNA sequence of the CRO binds DNMT and reduces methylating activity.As described herein, DNMT strongly interacts with RNA in a manner thatis not sequence-specific. In particular, DNMT can bind RNA more stronglythan DNA with the same primary structure. Optionally, binding betweenthe CRO and DNMT could be further enhanced by including one or moremodified nucleotides (e.g., cytidine or analogs thereof) that covalentlybind to DNMT. Exemplary cytidine analogs are described herein.

In addition to binding DNMT, the CRO includes a sequence to selectivelyor specifically hybridizing to its target sequence, i.e., anextra-coding RNA (ecRNA), of a gene. Binding of the CRO to the ecRNA canbe determined by any useful method. Optionally, the methods can beperformed under stringent conditions. For example, BLAST searches can beperformed at high stringency conditions (as shown in FIGS. 7B-7C) and/orNorthern blot hybridization assays can be performed at a lowerconcentration of sodium chloride (e.g., 0.2 M NaCl, 0.1 M NaCl, or 50 mMNaCl) (as shown in FIG. 7D). Exemplary gene targets for CROs areprovided herein.

Extra-Coding RNA

Extra-coding RNAs (ecRNAs) can include both coding and non-codingregions of a gene. In particular, the CRO includes a sequence that isgene-specific. Exemplary genes include: C/EBPa (CCAAT enhancer bindingprotein alpha, cluster C (GO Accession No.: GO:0010605), HGNC AccessionNo.: 1833, NCBI Ref. Nos.: NP_004355.2, NM_004364.3); SPI1 (spleen focusforming virus (SFFV) proviral integration oncogene spi1, cluster C(GO:0010605), HGNC:11241, NCBI: NP_001074016.1, NM_ 001080547.1); RXRA(retinoid X receptor, alpha, cluster C (GO:0010605), HGNC:10477,NCBI:NP_002948.1, NM_002957.4); RARB (retinoic acid receptor, beta,HGNC:9865, NCBI:NP_000956.2, NM_000965.3); RB1 (retinoblastoma 1,cluster C (GO:0007049, GO:0051276, GO:0022402, GO:0006325, GO:0000278,GO:0016568, GO:0010605, and GO:0022403), HGNC:9884, NCBI:NP_000312.2,NM_000321.2); CDKN2A (cyclin-dependent kinase inhibitor 2A, HGNC:1787,NCBI:NP_478102.1, NM_058195.2); CDH1 (cadherin 1, type 1, E-cadherin,HGNC:1748, NCBI:BAA88957.1, AB025106.1); CDH13 (cadherin 13, H-cadherin,HGNC:1753, NCBI:NP_001248.1, NM_001257.3); TIMP3 (TIMP metallopeptidaseinhibitor 3, HGNC:11822, NCBI:NP_000353.1, NM_000362.4); VHL (vonHippel-Lindau tumor suppressor, HGNC:12687, NCBI:NP_000542.1,NM_000551.2); MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2,HGNC:7127, NCBI:NP_000240.1, NM_000249.3); MGMT (O-6-methylguanine-DNAmethyltransferase, HGNC:7059, NCBI:NP_002403.2, NM_002412.3); BRCA1(breast cancer 1, early onset, HGNC:1100, NCBI:NP_009231.2,NM_007300.3); GSTP1 (glutathione S-transferase pi 1, HGNC:4638,NCBI:NP_000843.1, NM_000852.3); HLTF (helicase-like transcriptionfactor, HGNC:11099, NCBI:NP_620636.1, NM_139048.2); RASSF1 (Rasassociation (RalGDS/AF-6) domain family member 1, HGNC:9882,NCBI:NP_733833.1, NM_170715.1); SOCS1 (suppressor of cytokine signaling1, HGNC:19383, NCBI:NP_003736.1, NM_003745.1); ESR1 (estrogen receptor1, HGNC:3467, NCBI:NP_001116214.1, NM_001122742.1); and DAPK1(death-associated protein kinase 1, HGNC:2674, NCBI:NP_004929.2,NM_004938.2). Exemplary gene-specific sequences for the CRO include the3′-end of C/EBPa ecRNA provided in FIG. 8 (SEQ ID NO:1) and fragments ofthe C/EBPa ecRNA provided in FIG. 11 (SEQ ID N0:8); a region of SPI1ecRNA, including the four regions provided in FIG. 9 (SEQ ID NOs:2-5); aregion of RXRA ecRNA, including the two regions provided in FIG. 10 (SEQID NOs:6-7); and a region of RARB ecRNA, including fragments of the RARBecRNA provided in FIG. 12 (SEQ ID NO:9).

Based on the various genes described herein (e.g., any described herein,such as those in cluster C), the gene-specific sequences for the CRO canbe designed having a sequence that is at least 80% (e.g., at least 85%,90%, 95%, 96%, 97%, 98%, 99% or more) complementary to a region spanningthe promoter, the coding part of the gene, and/or the 3′-downstream part(e.g., −2 kilobases and/or +2 kilobases from the transcriptional startsite (TSS) or the transcriptional end site (TES) of the genes,respectively). Any useful method can be used to determine such TSS andTES positions, including various databases (e.g., TESS: TranscriptionElement Search System, available at www.cbil.upenn.edu/tess/, andTranscriptional Regulatory Element Database (TRED), available atrulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home). Exemplarygene-specific sequences for the CRO include the 3′-end of C/EBPa ecRNAprovided in FIG. 8 (SEQ ID NO:1) and fragments of the C/EBPa ecRNAprovided in FIG. 11 (SEQ ID N0:8); a region of SPI1 ecRNA, including thefour regions provided in FIG. 9 (SEQ ID NOs:2-5); a region of RXRAecRNA, including the two regions provided in FIG. 10 (SEQ ID NOs:6-7);and a region of RARB ecRNA, including fragments of the RARB ecRNAprovided in FIG. 12 (SEQ ID NO:9).

Also described herein are methods of identifying ecRNAs, as well as CROsthat bind to such ecRNAs to form a complex and such complexes that bindDNMT. In some non-limiting embodiments, the CRO includes a sequence thatis gene-specific (e.g., a complementary to a particular gene), where thegene is in cluster C, as described herein (e.g., see Example 8).

In some embodiments, the genes in cluster C belong to particular GeneOntology (GO) categories (www.geneontology.org and The Gene OntologyConsortium, “Gene ontology: tool for the unification of biology,” Nat.Genet. 25(1):25-29 (2009)). Exemplary non-limiting GO categories includegenes related to conversion of one or more primary RNA transcripts intoone or more mature RNA molecules (GO:0006396, RNA processing); genesrelated to biochemical and morphological phases that occur duringsuccessive cell or nuclear replication events (GO:0007049, cell cycle),genes related to chemical reactions and pathways involving mRNA(GO:0016071, mRNA metabolic process), genes related to assembly,arrangement, or disassembly of chromosomes (GO:0051276, chromosomeorganization), genes related to splicing and joining primary RNAtranscript to form mature form of the RNA (GO:0008380, RNA splicing),genes related to cellular breakdown of macromolecules, large molecules(e.g., proteins), nucleic acids and carbohydrates (GO:0044265, cellularmacromolecule catabolic process), genes related to cellular metabolicprocess involving deoxyribonucleic acid (GO:0006259, DNA metabolicprocess), genes related to conversion of a primary mRNA transcript intoone or more mature mRNA (GO:0006397, mRNA processing), genes relate tobreakdown of a macromolecule or any molecule of high relative molecularmass having multiple repetition of subunits (GO:0009057, macromoleculecatabolic process), genes related to cellular processes involved in theprogression of biochemical and morphological events that occur duringsuccessive cell or nuclear replication events (GO:0022402, cell cycleprocess), genes related to the specification, formation, or maintenanceof the physical structure of eukaryotic chromatin (GO:0006325, chromatinorganization), genes related to progression through the phases of themitotic cell cycle (GO:0000278, mitotic cell cycle), genes related toalteration of DNA, protein, or RNA in chromatin (GO:0016568, chromatinmodification), genes related to processes that decreases the frequency,rate, or extent of the chemical reactions and pathways involvingmacromolecules (GO:0010605, negative regulation of macromoleculemetabolic process), genes related to chemical reactions and pathwaysresulting in the breakdown of a protein either with or without thehydrolysis of peptide bonds (GO:0030163, protein catabolic process),genes related to chemical reactions and pathways resulting in thebreakdown of a protein or peptide (GO:0019941, modification-dependentprotein catabolic process), genes related to chemical reactions andpathways resulting in the breakdown of a macromolecule (GO:0043632,modification-dependent macromolecule catabolic process), genes relatedto hydrolysis of a peptide resulting in the breakdown of a protein(GO:0051603, proteolysis involved in cellular protein catabolicprocess), and genes related to cell cycle processes involved in theprogression of biochemical and morphological phases that occur duringsuccessive cell or nuclear replication events (GO:0022403, cell cyclephase), where definitions for such terms and related GO accessionsnumbers are provided on www.geneontology.org (AmiGO version 1.8, lastaccessed Apr. 11, 2012). Further exemplary non-limiting GO categoriesinclude GO:0044257, cellular protein catabolic process; GO:0006281, DNArepair; GO:0006974, response to DNA damage stimulus; GO:0006412,translation; GO:0006511, ubiquitin-dependent protein catabolic process;GO:0033554, cellular response to stress; GO:0046907, intracellulartransport; GO:0000375, RNA splicing, via transesterification reactions;GO:0000398, nuclear mRNA splicing, via spliceosome; GO:0000377, RNAsplicing, via transesterification reactions with bulged adenosine asnucleophile; GO:0051301, cell division; GO:0034660, ncRNA metabolicprocess; GO:0000279, M phase; GO:0010558, negative regulation ofmacromolecule biosynthetic process; GO:0010629, negative regulation ofgene expression; GO:0009890, negative regulation of biosyntheticprocess; GO:0006260, DNA replication; GO:0006350, transcription;GO:0045449, regulation of transcription; GO:0031327, negative regulationof cellular biosynthetic process; GO:0010498, proteasomal proteincatabolic process; GO:0043161, proteasomal ubiquitin-dependent proteincatabolic process; GO:0000280, nuclear division; GO:0007067, mitosis;GO:0048285, organelle fission; GO:0051726, regulation of cell cycle;GO:0000087, M phase of mitotic cell cycle; GO:0045934, negativeregulation of nucleobase, nucleoside, nucleotide and nucleic acidmetabolic process; GO:0043933, macromolecular complex subunitorganization; and GO:0016481, negative regulation of transcription.

Exemplary non-limiting genes in GO Accession No. GO:0006396 (RNAprocessing) include those having the following abbreviated name, thefull name, the HGNC Accession No., and the chromosome location: SCAF1(SR-related CTD-associated factor 1, HGNC:30403, 19q13.3-q13.4); RALY(RNA binding protein, autoantigenic (hnRNP-associated with lethal yellowhomolog (mouse)), HGNC:15921, 20q11.21-q11.23); NCBP2 (nuclear capbinding protein subunit 2, 20 kDa, HGNC:7659, 3q29); EIF2C2 (eukaryotictranslation initiation factor 2C, 2, HGNC:3263, 8q24); CHERP (calciumhomeostasis endoplasmic reticulum protein, HGNC:16930, 19p13.1); RPL14(ribosomal protein L14, HGNC:10305, 3p22-p21.2); RBM3 (RNA binding motif(RNP1, RRM) protein 3, HGNC:9900, Xp11.2); LSM6 (LSM6 homolog, U6 smallnuclear RNA associated (S. cerevisiae), HGNC:17017, 4q31.21); RBM4 (RNAbinding motif protein 4, HGNC:9901, 11q13); RBM5 (RNA binding motifprotein 5, HGNC:9902, 3p21.3); SYNCRIP (synaptotagmin binding,cytoplasmic RNA interacting protein, HGNC:16918, 6q14-q15); ZNF638 (zincfinger protein 638, HGNC:17894, 2p13.1); SART3 (squamous cell carcinomaantigen recognized by T cells 3, HGNC:16860, 12q24.11); WTAP (Wilmstumor 1 associated protein, HGNC:16846, 6q25-q27); PNN (pinin, desmosomeassociated protein, HGNC:9162, 14q21.1); PUS7L (pseudouridylate synthase7 homolog (S. cerevisiae)-like, HGNC:25276, 12q12); APP (amyloid beta(A4) precursor protein, HGNC:620, 21q21.2); DDX23 (DEAD(Asp-Glu-Ala-Asp) box polypeptide 23 (SEQ ID NO:56), HGNC:17347,12q13.11); DHX38 (DEAD (Asp-Glu-Ala-His) box polypeptide 38 (SEQ IDNO:57), HGNC:17211, 16q22); SRRM2 (serine/arginine repetitive matrix 2,HGNC:16639, 16p13.3); TARDBP (TAR DNA binding protein, HGNC:11571,1p36.22); INTS6 (integrator complex subunit 6, HGNC:14879, 13q14.3);U2AF1 (U2 small nuclear RNA auxiliary factor 1, HGNC:12453, 21q22.3);LSM5 (LSM5 homolog, U6 small nuclear RNA associated (S. cerevisiae),HGNC:17162, 7p14.3); LSM4 (LSM4 homolog, U6 small nuclear RNA associated(S. cerevisiae), HGNC:17259, 19p13.1); SRRM1 (serine/arginine repetitivematrix 1, HGNC:16638, 1p36); LSM3 (LSM3 homolog, U6 small nuclear RNAassociated (S. cerevisiae), HGNC:17874, 3p25.1); PTBP2 (polypyrimidinetract binding protein 2, HGNC:17662, 1p21.3); TGS1 (trimethylguanosinesynthase 1, HGNC:17843, 8q11); RBM10 (RNA binding motif protein 10,HGNC:9896, Xp11.3); IMP4 (IMP4, U3 small nucleolar ribonucleoprotein,homolog (yeast), HGNC:30856, 2q21.1); CCAR1 (cell division cycle andapoptosis regulator 1, HGNC:24236, 10q22.1); PABPN1 (poly(A) bindingprotein, nuclear 1, HGNC:8565, 14q11.2); RRP1 (ribosomal RNA processing1 homolog (S. cerevisiae), HGNC:18785, 21q22.3); SF3B14 (Splicing Factor3B, 14-kD subunit, HPRD: 09702, 2pter-p25.1); EMG1 (EMG1 nucleolarprotein homolog (S. cerevisiae), HGNC:16912, 12p13); PTBP1(polypyrimidine tract binding protein 1, HGNC:9583, 19p13.3); HNRNPA2B1(heterogeneous nuclear ribonucleoprotein A2/B1, HGNC:5033, 7p15); RRP9(ribosomal RNA processing 9, small subunit (SSU) processome component,homolog (yeast), HGNC:16829, 3p21.31); HNRNPR (heterogeneous nuclearribonucleoprotein R, HGNC:5047, 1p36.12); HNRNPU (heterogeneous nuclearribonucleoprotein U (scaffold attachment factor A), HGNC:5048, 1q44);RNMTL1 (RNA methyltransferase like 1, HGNC:18485, 17p13.3); BICD1(bicaudal D homolog 1 (Drosophila), HGNC:1049, 12p11.2-p11.1); RSL1D1(ribosomal L1 domain containing 1, HGNC:24534, 16p13.13); WDR83 (WDrepeat domain 83, HGNC:32672, 19p13.13); PCF11 (PCF11, cleavage andpolyadenylation factor subunit, homolog (S. cerevisiae), HGNC:30097,11q13); PA2G4 (proliferation-associated 2G4, 38 kDa, HGNC:8550,12q13.2); NOP2 (NOP2 nucleolar protein homolog (yeast), HGNC:7867,12p13); RPS16 (ribosomal protein S16, HGNC:10396, 19q13.1); RPS14(ribosomal protein S14, HGNC:10387, 5q31-q33); SNRPA (small nuclearribonucleoprotein polypeptide A, HGNC:11151, 19q13.1); SNRPG (smallnuclear ribonucleoprotein polypeptide G, HGNC11163, 2p13.3); CPSF3L(cleavage and polyadenylation specific factor 3-like, HGNC:26052,1p36.33); FIP1L1 (FIP1 like 1 (S. cerevisiae), HGNC:19124, 4q11-q12);ELAC2 (elaC homolog 2 (E. coli), HGNC:14198, 17p11.2); PUS1(pseudouridylate synthase 1, HGNC:15508, 12q24); STRAP (serine/threoninekinase receptor associated protein, HGNC:30796, 12p13.1); TRPT1 (tRNAphosphotransferase 1, HGNC:20316, 11q13); NAA38(N(alpha)-acetyltransferase 38, NatC auxiliary subunit, HGNC:20471,7q31.1-q31.3); ZFC3H1 (zinc finger, C3H1-type containing, HGNC:28328,12q21.1); HNRNPL (heterogeneous nuclear ribonucleoprotein L, HGNC:5045,19q13.2); HNRNPM (heterogeneous nuclear ribonucleoprotein M, HGNC:5046,19p13.3-p13.2); DDX46 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 (SEQ IDNO 56), HGNC:18681, 5q31.1); RPS28 (ribosomal protein S28, HGNC:10418,19p13.2); CNOT6L (CCR4-NOT transcription complex, subunit 6-like,HGNC:18042, 4q13.3); METTLI (methyltransferase like 1, HGNC:7030,12q13); HNRNPF (heterogeneous nuclear ribonucleoprotein F, HGNC:5039,10q11.21); PRKRA (protein kinase, interferon-inducible double strandedRNA dependent activator, HGNC:9438, 2q31.2); HNRNPD (heterogeneousnuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37kDa), HGNC:5036, 4q21); HNRNPC (heterogeneous nuclear ribonucleoproteinC (C1/C2), HGNC:5035, 14q11); RPL10A (ribosomal protein L10a,HGNC:10299, 6p21.31); PABPC1 (poly(A) binding protein, cytoplasmic 1,HGNC:8554, 8q22.2-q23); ARL6IP4 (ADP-ribosylation-like factor 6interacting protein 4, HGNC:18076, 12q24.31); DDX41 (DEAD(Asp-Glu-Ala-Asp) box polypeptide 41 (SEQ ID NO:56), HGNC:18674,5q35.3); RPS24 (ribosomal protein S24, HGNC:10411, 10q22); PRPF40A(PRP40 pre-mRNA processing factor 40 homolog A (S. cerevisiae),HGNC:16463, 2q23.3); RTCD1 (RICA) (RNA 3′-terminal phosphate cyclase,HGNC:17981, 1p13.3); MPHOSPH10 (M-phase phosphoprotein 10 (U3 smallnucleolar ribonucleoprotein), HGNC:7213, 2p13.3); SMAD2 (SMAD familymember 2, HGNC:6768, 18q21); RNPS1 (RNA binding protein S1, serine-richdomain, HGNC:10080, 16p13.3); CASC3 (cancer susceptibility candidate 3,HGNC:17040, 17q11-q21.3); INTS10 (integrator complex subunit 10,HGNC:25548, 8p21.3); DDX5 (DEAD (Asp-Glu-Ala-Asp) box helicase 5 (SEQ IDNO:56), HGNC:2746, 17q21); U2AF1L4 (U2 small nuclear RNA auxiliaryfactor 1-like 4, HGNC:23020, 19q13.13); RBMX (RNA binding motif protein,X-linked, HGNC:9910, Xq26); HNRNPA1 (heterogeneous nuclearribonucleoprotein A1, HGNC:5031, 12q13.1); HNRNPA0 (heterogeneousnuclear ribonucleoprotein A0, HGNC:5030, 5q31); NOP14 (NOP14 nucleolarprotein hornolog (yeast), HGNC:16821, 4p16.3); URM1 (ubiquitin relatedmodifier 1, HGNC:28378, 9q34.13); DDX56 (DEAD (Asp-Glu-Ala-Asp) boxhelicase 56 (SEQ ID NO:56), HGNC:18193, 7p13); HNRNPH3 (heterogeneousnuclear ribonucleoprotein H3 (2H9), HGNC:5043, 10q22); UPF3B (UPF3regulator of nonsense transcripts homolog B (yeast), HGNC:20439,Xq25-q26); NOLC1 (nucleolar and coiled-body phosphoprotein 1,HGNC:15608, 10q24.32); GTF2F1 (general transcription factor IIF,polypeptide 1, 74 kDa, HGNC:4652, 19p13.3); WDR3 (WD repeat domain 3,HGNC:12755, 1p12); DDX54 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (SEQID NO:56), HGNC:20084, 12q24.11); HNRNPH1 (heterogeneous nuclearribonucleoprotein H1 (H), HGNC:5041, 5q35.3); SMC1A (structuralmaintenance of chromosomes 1A, HGNC:11111, Xp11.22-p11.21); PDCD7(programmed cell death 7, HGNC:8767, 15q22.2); POP7 (processing ofprecursor 7, ribonuclease P/MRP subunit (S. cerevisiae), HGNC:19949,7q22); PUF60 (poly-U binding splicing factor 60KDa, HGNC:17042, 8q24.3);ADAR (adenosine deaminase, RNA-specific, HGNC:225, 1q21.3); UTP18 (UTP18small subunit (SSU) processome component homolog (yeast), HGNC:24274,17q21.33); WBP4 (WW domain binding protein 4 (formin binding protein21), HGNC:12739, 13q13.3); HNRPLL (heterogeneous nuclearribonucleoprotein L-like, HGNC:25127, 2p22); RNGTT (RNAguanylyltransferase and 5′-phosphatase, HGNC:10073, 6q16); DKC1(dyskeratosis congenita 1, dyskerin, HGNC:2890, Xq28); DGCR8 (DiGeorgesyndrome critical region gene 8, HGNC:2847, 22q11.2); TRMT6 (tRNAmethyltransferase 6 homolog (S. cerevisiae), HGNC:20900, 20p12.3); PCBP1(poly(rC) binding protein 1, HGNC:8647, 2p13-p12); PCBP2 (poly(rC)binding protein 2, HGNC:8648, 12q13.12-q13.13); QKI (QKI, KH domaincontaining, RNA binding, HGNC:21100, 6q26); TSEN2 (tRNA splicingendonuclease 2 homolog (S. cerevisiae), HGNC:28422, 3p25.2); FTSJ1 (FtsJhomolog 1 (E. coli), HGNC:13254, Xp11.23); ZCCHC6 (zinc finger, CCHCdomain containing 6, HGNC:25817, 9q21); DUS1L (dihydrouridine synthase1-like (S. cerevisiae), HGNC:30086, 17q25.3); FTSJ3 (FtsJ homolog 3 (E.coli), HGNC:17136, 17q23); TFIP11 (tuftelin interacting protein 11,HGNC:17165, 22q12.1); PPP2R1A (protein phosphatase 2, regulatory subunitA, alpha, HGNC:9302, 19q13); EXOSC8 (exosome component 8, HGNC:17035,13q13.1); ZCCHC11 (zinc finger, CCHC domain containing 11, HGNC:28981,1p32.3); EXOSC4 (exosome component 4, HGNC:18189, 8q24.3); SARS(seryl-tRNA synthetase, HGNC:10537, 1p13.3); SF1 (splicing factor 1,HGNC:12950, 11q13); PRPF3 (PRP3 pre-mRNA processing factor 3 homolog (S.cerevisiae), HGNC:17348, 1q21.1); EXOSCI (exosome component 1,HGNC:17286, 10q24); PRPF4 (PRP4 pre-mRNA processing factor 4 hornolog(yeast), HGNC:17349, 9q31-q33); TTF2 (transcription termination factor,RNA polymerase II, HGNC:12398, 1p13.1); PRPF6 (PRP6 pre-mRNA processingfactor 6 homolog (S. cerevisiae), HGNC:15860, 20q13.33); TARBP1 (TAR(HIV-1) RNA binding protein 1, HGNC:11568, 1q42.2); HNRPDL(heterogeneous nuclear ribonucleoprotein D-like, HGNC:5037, 4q21.22);ElF4A3 (eukaryotic translation initiation factor 4A3, HGNC:18683,17q25.3); PAPD4 (PAP associated domain containing 4, HGNC:26776,5q14.1); ROD1 (PTBP3) (polypyrimidine tract binding protein 3,HGNC:10253, 9q32); SNRNP200 (small nuclear ribonucleoprotein 200 kDa(U5), HGNC:30859, 2q11.2); CPSF7 (cleavage and polyadenylation specificfactor 7, 59 kDa, HGNC:30098, 11q12.2); CPSF6 (cleavage andpolyadenylation specific factor 6, 68 kDa, HGNC:13871, 12q15); SNRNP40(small nuclear ribonucleoprotein 40 kDa (U5), HGNC:30857, 1p35.2); THOC4(ALYREF) (Aly/REF export factor, HGNC:19071, 17q25.3); CPSF4 (cleavageand polyadenylation specific factor 4, 30 kDa, HGNC:2327, 7q22); RBM39(RNA binding motif protein 39, HGNC:15923, 20q11.22); CPSF3 (cleavageand polyadenylation specific factor 3, 73 kDa, HGNC:2326, 2p25.1); THOC3(THO complex 3, HGNC:19072, 5q35.3); CPSF2 (cleavage and polyadenylationspecific factor 2, 100 kDa, HGNC:2325, 14q31.1); NHP2 (NHP2ribonucleoprotein homolog (yeast), HGNC:14377, 5q35.3); TRUB2 (TruBpseudouridine (psi) synthase homolog 2 (E. coli), HGNC:17170, 9);PRPF38B (PRP38 pre-mRNA processing factor 38 (yeast) domain containingB, HGNC:25512, 1p13.3); SSU72 (SSU72 RNA polymerase II CTD phosphatasehornolog (S. cerevisiae), HGNC:25016, 1p36); THOC1 (THO complex 1,HGNC:19070, 18p11.32); POLR2H (polymerase (RNA) II (DNA directed)polypeptide H, HGNC:9195, 3q28); POLR2L (polymerase (RNA) II (DNAdirected) polypeptide L, 7.6 kDa, HGNC:9199, 11p15); POLR2J (polymerase(RNA) II (DNA directed) polypeptide J, 13.3 kDa, HGNC:9197, 7q11.2);TRA2B (transformer 2 beta homolog (Drosophila), HGNC:10781, 3q26.2-q27);UTP6 (UTP6, small subunit (SSU) processome component, homolog (yeast),HGNC:18279, 17q11.2); WBP11 (WW domain binding protein 11, HGNC:16461,12p12.3); QTRT1 (queuine tRNA-ribosyltransferase 1, HGNC:23797,19p13.3); POLR2D (polymerase (RNA) II (DNA directed) polypeptide D,HGNC:9191, 2q21); IVNS1ABP (influenza virus NSIA binding protein,HGNC:16951, 1q25.1-q31.1); POLR2C (polymerase (RNA) II (DNA directed)polypeptide C, 33 kDa, HGNC:9189, 16q13-q21); POLR2A (polymerase (RNA)II (DNA directed) polypeptide A, 220 kDa, HGNC:9187, 17p13.1); SF3B2(splicing factor 3b, subunit 2, 145 kDa, HGNC:10769, 11q13); PRPF19(PRP19/PSO4 pre-mRNA processing factor 19 homolog (S. cerevisiae),HGNC:17896, 11q12.2); EXOSC10 (exosome component 10, HGNC:9138,1p36.22); PPP2CA (protein phosphatase 2, catalytic subunit, alphaisozyme, HGNC:9299, 5q31.1); PRPF8 (PRP8 pre-mRNA processing factor 8homolog (S. cerevisiae), HGNC:17340, 17p13.3); USP39 (ubiquitin specificpeptidase 39, HGNC:20071, 2p11.2); ADAT2 (adenosine deaminase,tRNA-specific 2, HGNC:21172, 6q24.2); SCNM1 (sodium channel modifier 1,HGNC:23136, 1q21.3); SNRNP70 (small nuclear ribonucleoprotein 70 kDa(U1), HGNC:11150, 19q13.3); NSUN2 (NOP2/Sun domain family, member 2,HGNC:25994, 5p15.32); RBM28 (RNA binding motif protein 28, HGNC:21863,7q32.2); PPWD1 (peptidylprolyl isomerase domain and WD repeat containing1, HGNC:28954, 5q12.3); TYW1B (tRNA-yW synthesizing protein 1 homolog B(S. cerevisiae), HGNC:33908, 7q11.23); TXNL4B (thioredoxin-like 4B,HGNC:26041, 16q22.2); PDCD11 (programmed cell death 11, HGNC:13408,10q24.32); TRMU (tRNA 5-methylaminomethyl-2-thiouridylatemethyltransferase, HGNC:25481, 22q13); RBM23 (RNA binding motif protein23, HGNC:20155, 14q11.1); SF3A2 (splicing factor 3a, subunit 2, 66 kDa,HGNC:10766, 19p13.3-p13.2); SF3A1 (splicing factor 3a, subunit 1, 120kDa, HGNC:10765, 22q12.2); SF3A3 (splicing factor 3a, subunit 3, 60 kDa,HGNC:10767, 1p); SLBP (stem-loop binding protein, HGNC:10904, 4p16.3);DIS3 (DIS3 mitotic control homolog (S. cerevisiae), HGNC:20604,13q21.32); HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1,HGNC:17011, 19q13.31); PPP1R8 (protein phosphatase 1, regulatory subunit8, HGNC:9296, 1p35.3); SFPQ (splicing factor proline/glutamine-rich,HGNC:10774, 1p34.3); LSM10 (LSM10, U7 small nuclear RNA associated,HGNC:17562, 1p34.3); NOP56 (NOP56 ribonucleoprotein homolog (yeast),HGNC:15911, 20p13); RBM14 (RNA binding motif protein 14, HGNC:14219,11q13.2); and TXNL4A (thioredoxin-like 4A, HGNC:30551, 18q23).

Additional exemplary genes also include those having the followingabbreviated names, where the full name, the HGNC Accession No., and thechromosome location can be accessed by using any useful database (e.g.,HUGO Gene Nomenclature Committee (HGNC, available atwww.genenames.org/hgnc-searches), Human Protein Reference Database(HPRD, available at www.hprd.org/index_html), or the NCBI Database):GO:0007049, cell cycle (ADCY3, MRPL41, AURKB, CDC16, WTAP, CD2AP,CTNNB1, CDCA8, APP, DDX11, INCENP, ILK, VPS4B, CDCA2, VPS4A, H2AFX,TLK1, TLK2, SUPT5H, CCNA2, CDCA5, CCAR1, CDCA3, ANAPC1, RAN, LIG1, POLE,SKP2, LIG3, ESPL1, UBR2, LIG4, NCAPD3, DCTN2, NCAPD2, MAPK1, UHRF1, PPP1CA, RCC2, SPAG5, CNTROB, TBRG1, MAD2L2, MRE11A, CDC34, CALR, RCC1, PIN1,SAC3D1, NCAPG2, SPIN2B, TCF3, NUDC, SSSCA1, MLL, MKI67, MAP2K1, PCNT,NUF2, PCNP, CDC20, CDC26, RAD54L, ATM, NOTCH2, NOLC1, DMTF1, UBA3,CHAF1A, CCNT1, GTSE1, CCNE1, SEH1L, FANCI, DYNC1H1, FANCA, MYC, DHCR24,CDC7, CCNK, ARHGEF2, RBBP4, DSN1, CCNF, SF1, TPX2, PAPD7, RAD52, JMY,KLHDC3, RAD51, CCND1, CHMP1B, CCND3, CCND2, TOP3A, ADAM17, SIAH2, USP22,PML, BCCIP, ZNF655, NCAPH, NCAPG, BCL2, NPAT, PKD2, PPP3CA, BUB3,TRIP13, TXNL4B, UPF1, PDS5A, GMNN, ILF3, APPL1, MIS12, CCNB1, RASSF4,PSMD14, PSMD13, ERBB2IP, GSK3B, CALM3, CALM2, DNM2, TXNL4A, CALM1,BARD1, PRC1, PKMYT1, RBM7, MLL5, RAD21, TARDBP, TUBG1, ASPM, RBL2,PLKIS1, PPPICC, TACC3, PPP1CB, AHR, WEE1, PSMA2, PSMA1, PA2G4, EP300,TIMELESS, PSMA4, AKAP8, DSCC1, KATNB1, ANAPC11, PSMA7, CCNG2, PSMB5,PSMB4, GADD45GIP1, NIPBL, PSMB1, SMARCB1, FBXO43, TUBE1, CLASP2, HELLS,TFDP1, CREBL2, TAF1, WDR6, HDAC3, CDKN1B, PSMC5, PSMC4, PLK1, ZNF318,ABL1, SMCIA, CCNDBP1, MYH10, E2F1, MAD1L1, E2F2, E2F3, E2F4, E2F8, RHOU,AKT1, CDC45, MCM8, PSMC3IP, PSMD1, PSMD2, PSMD3, RANBP1, PSMD6, PSMD7,ARL2, NUSAP1, CDK6, UBE2I, RB1, MCM2, MCM3, CDK4, CDK5, CDK2, MCM6, GAK,MFN2, RIF1, PSME1, PSME2, UBC, HAUS5, FOXM1, USP9X, CAMK2G, HCFC1,CDC73, TSPYL2, HJURP, PAFAH1B1, GFI1, NFATC1, CiPS1, TXNIP, BOD1, MSH2,PSRC1, CENPJ, CDC25A, SMC4, GPS2, GSPT1, CUL4A, and SETD8); GO:0016071,mRNA metabolic process (SCAF1, RALY, NCBP2, EIF2C2, LSM6, RBM4, RBM5,SYNCRIP, WTAP, PNN, APP, DDX23, DNAJB11, DHX38, SRRM2, TARDBP, U2AF1,LSM5, SRRM1, LSM4, LSM3, PTBP2, RBM10, TGS1, CCAR1, PAN2, PABPN1, PAN3,SF3B14, HNRNPA2B1, PTBP1, HNRNPR, HNRNPU, WDR83, PCF11, SERBP1, VEGFA,SNRPA, EIF2C3, SNRPG, FIP1L1, STRAP, IGF2BP1, MAPKAPK2, NAA38, HNRNPL,HNRNPM, DDX46, CNOT6L, HNRNPF, HNRNPD, HNRNPC, PABPC1, DDX41, PRPF40A,SMG5, PAIP1, SMG7, SMG1, RNPS1, CASC3, DDX5, RBMX, HNRNPA1, U2AFIL4,HNRNPA0, UPF3A, HNRNPH3, UPF3B, PNRC2, GTF2F1, SMC1A, HNRNPH1, PUF60,ADAR, WBP4, HNRPLL, AUH, YBX2, RNGTT, PCBP1, PCBP2, QKI, TSEN2, TFIP11,SF1, PRPF3, PRPF4, TTF2, PRPF6, EIF4A3, PAPD4, ROD1, SNRNP200, CPSF7,CPSF6, THOC4, SNRNP40, CPSF4, RBM39, CPSF3, THOC3, CPSF2, PRPF38B,SSU72, THOC1, POLR2H, POLR2L, POLR2J, TRA2B, WBP11, POLR2D, POLR2C,SF3B2, POLR2A, EXOSC10, PRPF19, ZFP36L2, PRPF8, USP39, SCNM1, SNRNP70,RBM28, PPWD1, TXNL4B, RBM23, UPF1, SF3A2, SF3A1, SF3A3, SLBP, GSPT1,HNRNPUL1, PPP1R8, SFPQ, LSM10, RBM14, and TXNL4A); GO:0051276,chromosome organization (MORF4L1, HIST2H2AA3, HIST2H2AA4, HMGN2, RBM4,CBX4, CBX3, H1FX, KDM1A, BRPF1, CDCA8, MLL5, H2AFV, BRPF3, DDX11,SMARCD2, H2AFZ, H2AFY, H2AFX, TLK1, HIRIP3, TLK2, MLL3, SUPT5H, CDCA5,MLL2, SATB1, RBL2, C11ORF30, MTA2, RCOR1, ARID1A, ESPL1, ARID1B, LIG4,NCAPD3, NCAPD2, SUZ12, EP300, KDM2A, BAZ1B, SMARCE1, SMARCA5, AKAP8,SMARCA2, EP400, DSCC1, SUPT6H, HMGB1, HMGB2, TADA3, SETD1B, MRE11A,SETD1A, TRRAP, VPS72, NIPBL, ACD, SMARCB1, NCAPG2, RNF168, SUPT7L,ASF1B, BCOR, HELLS, HIST2H3A, AEBP2, EHMT1, SETDB2, SMCHD1, MLL, NFE2,CREBBP, WRN, KAT5, RAD54L, HIST2H3C, UIMC1, HDAC5, TNKS1BP1, MSL3,HDAC3, HDAC2, PHF1, MSL1, SMARCC1, SMARCC2, SUPT16H, DNMT1, H3F3B,CHAF1A, SMC1A, HDAC7, RERE, PTGES3, HP1BP3, ARID4B, EZH2, CTCF, DMAP1,DKC1, SEH1L, GPX4, HIST2H2AC, KDM5A, KDM5B, KDM5C, BRD8, UBE2A,HIST1H1E, RBBP4, PAPD7, NUSAP1, RB1, MCM2, RBBP7, HMGA1, C20ORF20, BPTF,HIST2H2BE, HIST2H2BF, USP21, RUVBL2, USP22, CARM1, SRCAP, KDM6B, PRKDC,CDC73, NR3C1, ARID2, NCAPH, TSPYL2, SET, CHD7, HJURP, NCAPG, SAFB, CHD1,KDM3A, SUPT4H1, CHD6, BAZ2A, TINF2, BUB3, CHD3, KAT2A, MSH3, HIST1H2BF,MSH2, SIRT1, MIS12, SMC4, DOT1L, WHSC1L1, HIST1H2AI, CABIN1, PHF21A,SETD7, SETD8, KDM4A, HIST1H2AM, SETD2, and RBM14); GO:0008380, RNAsplicing (SCAF1, RALY, NCBP2, LSM6, RBM4, RBM5, SYNCRIP, ZNF638, WTAP,PNN, DHX38, DDX23, SRRM2, TARDBP, U2AF1, LSM5, SRRM1, LSM4, LSM3, PTBP2,RBM10, TGS1, CCAR1, PABPN1, SF3B14, HNRNPA2B1, PTBP1, HNRNPR, HNRNPU,WDR83, PCF11, SNRPA, SNRPG, STRAP, TRPT1, NAA38, HNRNPL, HNRNPM, DDX46,HNRNPF, HNRNPD, HNRNPC, PABPC1, DDX41, ARL6IP4, PRPF40A, MPHOSPH10,RNPS1, CASC3, DDX5, RBMX, HNRNPA1, U2AF1L4, HNRNPA0, HNRNPH3, UPF3B,GTF2F1, SMC1A, HNRNPH1, PUF60, PDCD7, WBP4, PCBP1, PCBP2, QKI, TSEN2,TFIP11, PPP2R1A, SF1, PRPF3, PRPF4, TTF2, PRPF6, EIF4A3, SNRNP200,CPSF7, THOC4, SNRNP40, RBM39, CPSF3, CPSF2, THOC3, PRPF38B, THOC1,POLR2H, POLR2L, POLR2J, TRA2B, WBP11, POLR2D, IVNS1ABP, POLR2C, SF3B2,POLR2A, PRPF19, PRPF8, PPP2CA, USP39, SCNM1, SNRNP70, RBM28, PPWD1,TXNL4B, SF3A2, SF3A1, SF3A3, HNRNPUL1, PPP1R8, SFPQ, LSM10, RBM14, andTXNL4A); GO:0044265, cellular macromolecule catabolic process (MYLIP,CDC16, CD2AP, ZNRF2, WWP2, CUL9, CDCA3, AUP1, PAN2, ANAPC1, PAN3, SKP2,UBR4, UBR2, UBE2J2, UHRF1, PIAS4, FBXL8, UBR5, FBXL5, FBXL4, PIAS2,EIF2C3, FBXL3, RAD23B, RAD23A, UBA5, CDC34, ARIH1, ARIH2, FBXO9, FBXO7,PCNP, CDC20, CDC26, ATM, UPF3A, RNF6, UPF3B, UBA1, UBR2, UBA3, KIAA0368,LDLR, UBE2G1, UBE2G2, AUH, SENP6, FBXL19, FANCL, USP19, LONP1, MGRN1,NSMCE2, USP11, USP10, CCNO, MYC, USP14, ZCCHC11, UFD1L, HERC5, GTF2H3,HERC4, ERLIN1, HERC3, HERC2, WDR48, EIF4A3, SENP3, USP21, ADAM17, SIAH2,USP22, USP24, CLN6, USP7, USP3, C10ORF46, USP4, EDEM3, C12ORF51, EDEM1,MYCBP2, ZFP36L2, SUMO2, SQSTM1, RNASET2, USP39, PPP2CB, USP36, FBXW11,USP33, TRIP12, BUB3, USP31, UBXN1, UPF1, LRRC41, SPSB2, BIRC6, SOD1,CCNB1, PSMD14, PSMD13, PPP1R8, TCEB2, BARD1, NCBP2, IDE, CBX4, RABGEF1,RBCK1, RNF149, RNF34, RELA, USP1, SOCS7, PSMA2, PSMA1, MIB1, KDM2A,TRIM33, PSMA4, ASB1, RNF138, TNFAIP3, ASB6, NEDD8, ANAPC11, UBAC1,PSMA7, PSMB5, PSMB4, UBE2D2, FBXW5, PSMB1, FBXW4, FBXO43, HNRNPD,STAMBPL1, RNF168, RNF167, TRAF7, HECTD1, SMG5, SMG7, HACE1, SMG1, RNPS1,CASC3, ATG3, ATE1, URM1, PSMC5, PSMC4, VCP, OTUB1, PNRC2, BAP1, OS9,CASP3, PSMD1, PSMD2, PSMD3, PSMD6, PSMD7, UBE2A, DDB1, UBE2I, PSME1,UBE2K, PSME2, UBE2M, DDB2, UBC, KLHL12, UBE2S, CUEDC2, OTUD5, APH1A,UBE3B, USP9X, STUB1, UBE2R2, EXOSC10, RPA2, RNF123, ERCC5, MAP3K1,ERCC3, ERCC2, STAMBP, CBL, UBE2Q2, UBE2Q1, NCSTN, WSB1, MPG, HSP90B1,GMCL1, GSPT1, CUL4A, BAX, ENDOG, APAF1, and TBLIX); GO:0006259, DNAmetabolic process (MMS19, MORF4L1, XRCC3, RBM4, XRCC1, MLL5, RAD21,PSIP1, H2AFX, PMS1, CIB1, MCM3AP, POLK, POLI, POLH, POLG, RAN, C11ORF30,USP1, LIG1, POLE, LIG3, RAD9A, TOPBP1, LIG4, RMI1, RFC5, UHRF1, RPAIN,DCLRE1B, RFC2, TBRG1, TNFAIP1, DSCC1, RAD23B, HMGB1, HMGB2, RAD23A,MRE11A, CDC34, BANF1, TK1, ACD, POLE3, SMARCB1, RNF168, POLQ, HELLS,MLL, EME2, TP53BP1, EME1, BRIP1, SMG1, WRN, KAT5, RAD54L, UIMC1, ATM,TNKS1BP1, VCP, PPIA, SUPT16H, DNMT1, PARP4, HSPD1, KCTD13, CHAF1A, ABL1,SMC1A, DUT, PTGES3, UVRAG, ZNF12, CTCF, DMAP1, PMS2L2, FANCL, MCM9,ANKRD17, CDC45, LONP1, CASP3, MCM8, DKC1, NT5M, FANCI, PSMC3IP, NSMCE2,POLG2, FANCG, TOP2B, FANCA, CCNO, MYC, HEMK1, CDC7, UBE2A, RBBP4, REV1,CCDC88A, GEN1, DDB1, PAPD7, GTF2H3, MCM2, TOP1MT, RAD52, MCM3, RBBP7,MCM4, HMGA1, CDK2, MCM5, JMY, MCM6, KLHDC3, RAD51, RRM2, TOP3A, DDB2,RUVBL2, REV3L, WRNIP1, PML, PRKDC, BCCIP, POLA2, PRPF19, TYMS, RPA2,ERCC5, SET, MUS81, GATAD2A, ERCC3, BAZ2A, APEX1, TINF2, ERCC2, TRIP13,SSRP1, UPF1, MSH3, MSH2, SMC6, SOD1, SIRT1, CDC25A, MPG, CUL4A, CSNK1D,CSNK1E, BAX, ENDOG, SFPQ, APAF1, NFIC, RBM14, REPIN1, and BARD1);GO:0006397, mRNA processing (SCAF1, RALY, NCBP2, EIF2C2, LSM6, RBM4,RBM5, SYNCRIP, WTAP, PNN, APP, DHX38, DDX23, SRRM2, TARDBP, U2AF1, LSM5,SRRM1, LSM4, LSM3, PTBP2, RBM10, TGS1, CCAR1, PABPN1, SF3B14, HNRNPA2B1,PTBP1, HNRNPR, HNRNPU, WDR83, PCF11, SNRPA, SNRPG, FIP1L1, STRAP, NAA38,HNRNPL, HNRNPM, DDX46, CNOT6L, HNRNPF, HNRNPD, HNRNPC, PABPC1, DDX41,PRPF40A, RNPS1, CASC3, DDX5, HNRNPA1, RBMX, U2AF1L4, HNRNPA0, HNRNPH3,UPF3B, GTF2F1, SMC1A, HNRNPH1, PUF60, ADAR, WBP4, HNRPLL, RNGTT, PCBP1,PCBP2, QKI, TSEN2, TFIP11, SF1, PRPF3, PRPF4, TTF2, PRPF6, EIF4A3,PAPD4, ROD1, SNRNP200, CPSF7, CPSF6, THOC4, SNRNP40, CPSF4, RBM39,CPSF3, CPSF2, THOC3, PRPF38B, SSU72, THOC1, POLR2H, POLR2L, POLR2J,TRA2B, WBP11, POLR2D, POLR2C, SF3B2, POLR2A, PRPF19, PRPF8, USP39,SCNM1, SNRNP70, RBM28, PPWD1, TXNL4B, RBM23, SF3A2, SF3A1, SF3A3, SLBP,HNRNPUL1, PPP1R8, SFPQ, LSM10, RBM14, and TXNL4A); GO:0009057,macromolecule catabolic process (USE1, MYLIP, CDC16, CD2AP, ZNRF2, WWP2,CUL9, CDCA3, AUP1, PAN2, ANAPC1, PANS, SKP2, UBR4, UBR2, UBE2J2, UHRF1,PIAS4, FBXL8, UBR5, FBXL5, FBXL4, YME1L1, PIAS2, EIF2C3, FBXL3, RAD23B,RAD23A, UBA5, CDC34, ARIH1, ARIH2, FBXO9, FBXO7, GUSB, PCNP, CDC20,CDC26, AFG3L2, ATM, UPF3A, RNF6, UPF3B, UBA1, UBA2, UBA3, KIAA0368,LDLR, UBE2G1, UBE2G2, AUH, SENP6, FBXL19, FANCL, USP19, LONP1, MGRN1,NSMCE2, USP11, USP10, CCNO, MYC, USP14, DHCR24, ZCCHC11, UFD1L, HERC5,GTF2H3, HERC4, ERLIN1, HERC3, HERC2, WDR48, EIF4A3, SENP3, USP21, MGEA5,ADAM17, SIAH2, USP22, USP24, CLN6, USP7, C10ORF46, USP3, USP4, EDEM3,C12ORF51, EDEM1, MYCBP2, ZFP36L2, SUMO2, SQSTM1, RNASET2, USP39, PPP2CB,USP36, FBXW11, USP33, TRIP12, BUB3, USP31, UBXN1, UPF1, LRRC41, SPSB2,BIRC6, SOD1, CCNB1, PSMD14, PSMD13, PPP1R8, TCEB2, BARD1, NCBP2, IDE,CBX4, RABGEF1, RBCK1, RNF149, RNF34, USP1, RELA, SOCS7, PSMA2, PSMA1,MIB1, KDM2A, TRIM33, PSMA4, ASB1, RNF138, TNFAIP3, ASB6, NEDD8, ANAPC11,PSMA7, UBAC1, PSMB5, PSMB4, UBE2D2, FBXW5, PSMB1, FBXW4, FBXO43,STAMBPL1, HNRNPD, RNF168, RNF167, TRAF7, HECTD1, SMG5, SMG7, HACE1,SMG1, RNPS1, CASC3, ATG3, ATE1, URM1, PSMC5, PSMC4, VCP, OTUB1, PNRC2,BAP1, OS9, AKT1, CASP3, PSMD1, PSMD2, PSMD3, PSMD6, MAN2B1, PSMD7,UBE2A, DDB1, UBE2I, CDK5, PSME1, PSME2, UBE2K, UBE2M, DDB2, UBC, KLHL12,UBE2S, CUEDC2, OTUD5, APH1A, UBE3B, USP9X, STUB1, UBE2R2, NGLY1,EXOSC10, RPA2, RNF123, ERCC5, MAP3K1, ERCC3, ERCC2, STAMBP, CBL, UBE2Q2,UBE2Q1, NCSTN, WSB1, MPG, HSP90B1, GMCL1, GSPT1, CUL4A, BAX, ENDOG,APAF1, and TBL1X); GO:0022402, cell cycle process (ADCY3, PRC1, PKMYT1,RBM7, AURKB, CDC16, CD2AP, CTNNB1, CDCA8, APP, MLL5, RAD21, DDX11,INCENP, TARDBP, ILK, CDCA2, H2AFX, TUBG1, CCNA2, CDCA5, ASPM, CDCA3,ANAPC1, RAN, POLE, LIG3, SKP2, UBR2, ESPL1, PLK1S1, TACC3, PPP1CB, WEE1,NCAPD3, NCAPD2, DCTN2, PSMA2, PSMA1, PA2G4, RCC2, TIMELESS, SPAG5,PSMA4, CNTROB, TBRG1, AKAP8, MAD2L2, DSCC1, MRE11A, KATNB1, ANAPC11,CDC34, CALR, PSMA7, RCC1, CCNG2, PSMB5, PSMB4, NIPBL, PSMB1, SAC3D1,NCAPG2, FBXO43, TUBE1, CLASP2, TCF3, HELLS, NUDC, SSSCA1, TAF1, MKI67,MAP2K1, PCNT, WDR6, NUF2, CDC20, CDC26, RAD54L, ATM, NOTCH2, HDAC3,CDKN1B, PSMC5, PSMC4, NOLC1, PLK1, ZNF318, ABL1, SMC1A, MYH10, E2F1,MAD1L1, E2F4, RHOU, GTSE1, AKT1, CCNE1, SEH1L, PSMC3IP, PSMD1, PSMD2,PSMD3, RANBP1, PSMD6, DYNC1H1, FANCA, PSMD7, MYC, DHCR24, CDC7, CCNK,ARHGEF2, DSN1, CCNF, SF1, PAPD7, TPX2, NUSAP1, UBE2I, CDK6, RB1, RAD52,CDK4, CDK2, JMY, KLHDC3, RAD51, MFN2, CCND1, PSME1, CCND2, PSME2, UBC,TOP3A, ADAM17, HAUS5, USP9X, CAMK2G, PML, ZNF655, NCAPH, NCAPG, BCL2,NPAT, PKD2, PAFAH1B1, GFI1, PPP3CA, BUB3, TRIP13, NFATC1, TXNL4B, BOD1,PDS5A, MSH2, ILF3, CENPJ, CDC25A, MIS12, SMC4, CCNB1, PSMD14, PSMD13,GSPT1, CUL4A, GSK3B, SETD8, DNM2, TXNL4A, and BARD1); GO:0006325,chromatin organization (MORF4L1, HIST2H2AA3, HIST2H2AA4, HMGN2, RBM4,CBX4, CBX3, H1FX, KDM1A, MLL5, BRPF1, H2AFV, BRPF3, SMARCD2, H2AFZ,H2AFY, H2AFX, TLK1, HIRIP3, TLK2, MLL3, SUPT5H, MLL2, SATB1, RBL2,C11ORF30, RCOR1, MTA2, ARID1A, ARID1B, SUZ12, EP300, KDM2A, BAZ1B,SMARCE1, SMARCA5, SMARCA2, EP400, SUPT6H, HMGB1, HMGB2, TADA3, SETD1B,SETD1A, TRRAP, VPS72, SMARCB1, RNF168, SUPT7L, ASF1B, BCOR, HELLS,HIST2H3A, AEBP2, MLL, SETDB2, NFE2, EHMT1, CREBBP, KAT5, HIST2H3C,UIMC1, HDAC5, MSL3, HDAC3, HDAC2, PHF1, MSL1, SMARCC1, SMARCC2, SUPT16H,DNMT1, H3F3B, CHAF1A, HDAC7, RERE, HP1BP3, ARID4B, EZH2, CTCF, DMAP1,GPX4, HIST2H2AC, KDM5A, KDM5B, KDM5C, BRD8, HIST1H1E, UBE2A, RBBP4, RB1,MCM2, RBBP7, HMGA1, C20ORF20, BPTF, HIST2H2BE, HIST2H2BF, USP21, RUVBL2,USP22, CARM1, SRCAP, KDM6B, CDC73, NR3C1, ARID2, TSPYL2, SET, CHD7,HJURP, SAFB, CHD1, KDM3A, SUPT4H1, CHD6, BAZ2A, CHD3, KAT2A, H1ST1H2BF,SIRT1, DOT1L, WHSC1L1, HIST1H2AI, CABIN1, PHF21A, SETD7, SETD8, KDM4A,HIST1H2AM, SETD2, and RBM14); 60:0000278, mitotic cell cycle (PRC1,PKMYT1, AURKB, CDC16, CD2AP, CDCA8, APP, RAD21, DDX11, INCENP, TARDBP,CDCA2, CCNA2, CDCA5, ASPM, CDCA3, ANAPC1, RAN, POLE, SKP2, ESPL1,PPP1CB, WEE1, NCAPD3, NCAPD2, DCTN2, PSMA2, PSMA1, TIMELESS, RCC2,SPAG5, PSMA4, AKAP8, MAD2L2, DSCC1, KATNB1, ANAPC11, CDC34, PSMA7, RCC1,CCNG2, PSMB5, PSMB4, NIPBL, PSMB1, SAC3D1, NCAPG2, CLASP2, TCF3, HELLS,NUDC, SSSCA1, TAF1, MAP2K1, NUF2, CDC20, CDC26, CDKN1B, PSMC5, NOLC1,PSMC4, PLK1, UBA3, SMC1A, ABL1, E2F1, MAD1L1, E2F4, RHOU, GTSE1, AKT1,CCNE1, SEHIL, PSMD1, PSMD2, PSMD3, PSMD6, DYNC1H1, PSMD7, CDC7, ARHGEF2,CCNK, DSN1, CCNF, PAPD7, TPX2, NUSAP1, UBE2I, CDK6, RB1, CDK4, CDK2,CCND1, PSME1, CCND2, PSME2, UBC, ADAM17, HAUS5, CAMK2G, USP9X, NCAPH,NCAPG, BCL2, NPAT, PAFAH1B1, GFI1, PPP3CA, BUB3, NFATC1, BOD1, TXNL4B,PDS5A, MIS12, CDC25A, SMC4, CCNB1, PSMD14, PSMD13, CUL4A, GSPT1, SETD8,TXNL4A, and DNM2); GO:0016568, chromatin modification (MORF4L1, RBM4,CBX4, CBX3, KDM1A, MLL5, BRPF1, SMARCD2, BRPF3, H2AFY, TLK1, TLK2,SUPT5H, MLL3, MLL2, RBL2, C11ORF30, RCOR1, ARID1A, ARID1B, SUZ12, EP300,SMARCE1, BAZ1B, KDM2A, SMARCA5, SMARCA2, EP400, SUPT6H, TADA3, SETD1B,SETD1A, TRRAP, VPS72, SMARCB1, SUPT7L, RNF168, BCOR, ASF1B, HELLS,AEBP2, MLL, SETDB2, EHMT1, CREBBP, KAT5, UIMC1, MSL3, HDAC5, HDAC3,HDAC2, PHF1, SMARCC1, MSL1, SMARCC2, DNMT1, RERE, HDAC7, EZH2, CTCF,DMAP1, KDM5A, KDM5B, KDM5C, BRD8, UBE2A, RBBP4, RB1, RBBP7, C20ORF20,BPTF, USP21, RUVBL2, USP22, CARM1, SRCAP, KDM6B, CDC73, NR3C1, ARID2,TSPYL2, CHD7, HJURP, CHD1, KDM3A, SUPT4H1, CHD6, BAZ2A, CHD3, KAT2A,SIRT1, DOT1L, WHSC1L1, CAB1N1, SETD7, PHF21A, SETD8, KDM4A, RBM14, andSETD2); GO:0010605, negative regulation of macromolecule metabolicprocess (EIF2C2, CDC16, CITED2, CTNNB1, ZGPAT, SIN3A, WWP2, SUPT5H,EIF2B4, EIF2B5, ANAPC1, RXRA, SKP2, RAD9A, PIAS4, RPS14, LRCH4, EIF2C3,EIF2AK4, IGF2BP1, IGF2BP3, CALR, VPS72, ACD, ATN1, DRAP1, BCOR, TCF25,HNRNPAB, IKZF1, SMAD4, NDFIP2, SMAD2, CDC20, SKI, CDC26, UBP1, MLX,UBA3, NCOR2, ADAR, SPI1, ZEB2, DMAP1, HSBP1, YBX2, AES, NR2F2, FAM129A,MYC, ZNF496, CTBP1, ZCCHC11, ANKHD1-EIF4EBP3, SPEN, MBD2, RBBP7, MBD1,MXD3, EIF4A3, G6PD, MGEA5, MTDH, PML, SET, PPP2CA, GATAD2A, AATF,SUPT4H1, BAZ2A, BUB3, JARID2, GMNN, TRIM28, YWHAB, ILF3, ANKHD1, PHF12,EIF2B1, STAT3, PSMD14, PSMD13, GSK3A, YWHAQ, PHF21A, RBM15, BARD1, JDP2,THRA, XPO5, CCDC85B, RBM3, IDE, CBX4, CBX3, MAF1, KDM1A, FNTA, EIF4EBP2,SND1, TIA1, INSR, AKIRIN2, SATB1, RELA, MTA2, CSDA, PSMA2, SUZ12, PSMA1,PA2G4, SMARCE1, TIMELESS, TRIM33, PSMA4, ASB1, SMARCA2, HMGB1, HMGB2,TH1L, ANAPC11, PSMA7, WT1, PSMB5, PSMB4, PSMB1, ITGAV, PRKRA, RUNX2,HELLS, EHMT1, RFX5, KAT5, FOXP4, UIMC1, HDAC5, HDAC3, HDAC2, PSMC5,CDKN1B, PSMC4, SMARCC2, DNMT1, SCMH1, E2F1, HSP90AB1, FOXK1, CTCF,ZNF202, PDCD4, RPS3, CDC45, DGCR8, PCGF6, PSMD1, PSMD2, PSMD3, PSMD6,KDM5B, PSMD7, PPP2R1A, LDB1, UBE2I, RB1, PRKCD, CDK5, COBRA1, NCOA2,BPTF, PSME1, PSME2, UBC, PEBP1, CUX1, KDM6B, BTAF1, BCLAF1, TSPYL2,GFI1, TINF2, TNRC6A, ENO1, CEBPA, ZBTB7A, NACC1, MSH3, MSH2, SIRT5,CBY1, SIRT1, GPS2, SIRT3, DR1, PHB2, BAX, SETD8, VPS28, NFIC, andTBL1X); GO:0030163, protein catabolic process (IDE, USE1, CBX4, MYLIP,CDC16, CD2AP, ZNRF2, WWP2, CUL9, RABGEF1, RBCK1, RNF149, RNF34, AUP1,CDCA3, ANAPC1, PAN2, USP1, RELA, SKP2, UBR4, SOCS7, UBR2, UBE2J2, PSMA2,MIB1, PSMA1, UHRF1, KDM2A, PIAS4, TRIM33, FBXL8, UBR5, PSMA4, ASB1,FBXL5, RNF138, FBXL4, PIAS2, YME1L1, TNFAIP3, FBXL3, ASB6, RAD23B,RAD23A, NEDD8, UBA5, ANAPC11, CDC34, PSMA7, UBAC1, PSMB5, ARIH1, PSMB4,ARIH2, UBE2D2, PSMB1, FBXW5, FBXW4, FBXO43, STAMBPL1, RNF168, TRAF7,RNF167, FBXO9, HECTD1, FBXO7, HACE1, PCNP, CDC20, CDC26, AFG3L2, ATG3,ATE1, URM1, RNF6, PSMC5, PSMC4, VCP, UBA1, OTUB1, UBA2, UBA3, KIAA0368,UBE2G1, UBE2G2, BAP1, OS9, SENP6, FBXL19, FANCL, AKT1, USP19, LONP1,MGRN1, PSMD1, PSMD2, PSMD3, USP11, NSMCE2, USP10, PSMD6, PSMD7, USP14,UBE2A, UFD1L, DDB1, HERC5, HERC4, UBE2I, ERLIN1, HERC3, HERC2, WDR48,SENP3, PSME1, UBE2K, PSME2, UBE2M, UBC, DDB2, USP21, ADAM17, KLHL12,SIAH2, USP22, USP24, UBE2S, CLN6, USP7, CUEDC2, OTUD5, APH1A, USP3,UBE3B, C10ORF46, USP9X, USP4, EDEM3, C12ORF51, STUB1, EDEM1, MYCBP2,UBE2R2, SUMO2, RNF123, SQSTM1, MAP3K1, USP39, PPP2CB, USP36, FBXW11,USP33, TRIP12, BUB3, USP31, UBXN1, STAMBP, LRRC41, SPSB2, CBL, BIRC6,UBE2Q2, UBE2Q1, NCSTN, CCNB1, WSB1, PSMD14, HSP90B1, GMCL1, PSMD13,CUL4A, TCEB2, TBL1X, and BARD1); GO:0019941, modification-dependentprotein catabolic process (CBX4, MYLIP, CDC16, CD2AP, ZNRF2, WWP2, CUL9,RABGEF1, RBCK1, RNF149, RNF34, AUP1, CDCA3, PAN2, ANAPC1, USP1, UBR4,SKP2, SOCS7, UBR2, UBE2J2, PSMA2, MIB1, PSMA1, UHRF1, KDM2A, PIAS4,TRIM33, FBXL8, UBR5, PSMA4, FBXL5, ASB1, RNF138, FBXL4, PIAS2, TNFAIP3,FBXL3, ASB6, RAD23B, RAD23A, NEDD8, UBA5, ANAPC11, CDC34, PSMA7, UBAC1,PSMB5, ARIH1, PSMB4, ARIH2, UBE2D2, PSMB1, FBXW5, FBXW4, FBXO43,STAMBPL1, RNF168, TRAF7, RNF167, FBXO9, HECTD1, FBXO7, HACE1, PCNP,CDC20, CDC26, ATG3, ATE1, URM1, RNF6, PSMC5, PSMC4, VCP, UBA1, OTUB1,UBA2, UBA3, KIAA0368, UBE2G1, UBE2G2, BAP1, OS9, SENP6, FBXL19, FANCL,USP19, LONP1, MGRN1, PSMD1, PSMD2, PSMD3, USP11, NSMCE2, USP10, PSMD6,PSMD7, USP14, UBE2A, UFD1L, DDB1, HERC5, HERC4, UBE2I, ERLIN1, HERC3,HERC2, WDR48, SENP3, PSME1, UBE2K, PSME2, UBE2M, UBC, DDB2, USP21,KLHL12, SIAH2, USP22, USP24, UBE2S, USP7, CUEDC2, OTUD5, USP3, UBE3B,C10ORF46, USP9X, USP4, EDEM3, C12ORF51, STUB1, EDEM1, MYCBP2, UBE2R2,SUMO2, RNF123, SQSTM1, MAP3K1, USP39, PPP2CB, USP36, FBXW11, USP33,TRIP12, BUB3, USP31, UBXN1, STAMBP, LRRC41, CBL, SPSB2, BIRC6, UBE2Q2,UBE2Q1, CCNB1, WSB1, PSMD14, HSP90B1, GMCL1, PSMD13, CUL4A, TCEB2,TBL1X, and BARD1); GO:0043632, modification-dependent macromoleculecatabolic process (CBX4, MYLIP, CDC16, CD2AP, ZNRF2, WWP2, CUL9,RABGEF1, RBCK1, RNF149, RNF34, AUP1, CDCA3, PAN2, ANAPC1, USP1, UBR4,SKP2, SOCS7, UBR2, UBE2J2, PSMA2, MIB1, PSMA1, UHRF1, KDM2A, PIAS4,TRIM33, FBXL8, UBR5, PSMA4, FBXL5, ASB1, RNF138, FBXL4, PIAS2, TNFAIP3,FBXL3, ASB6, RAD23B, RAD23A, NEDD8, UBA5, ANAPC11, CDC34, PSMA7, UBAC1,PSMB5, ARIH1, PSMB4, ARIH2, UBE2D2, PSMB1, FBXW5, FBXW4, FBXO43,STAMBPL1, RNF168, TRAF7, RNF167, FBXO9, HECTD1, FBXO7, HACE1, PCNP,CDC20, CDC26, ATG3, ATE1, URM1, RNF6, PSMC5, PSMC4, VCP, UBA1, OTUB1,UBA2, UBA3, KIAA0368, UBE2G1, UBE2G2, BAP1, OS9, SENP6, FBXL19, FANCL,USP19, LONP1, MGRN1, PSMD1, PSMD2, PSMD3, USP11, NSMCE2, USP10, PSMD6,PSMD7, USP14, UBE2A, UFDIL, DDB1, HERC5, HERC4, UBE2I, ERLIN1, HERC3,HERC2, WDR48, SENP3, PSME1, UBE2K, PSME2, UBE2M, UBC, DDB2, USP21,KLHL12, SIAH2, USP22, USP24, UBE2S, USP7, CUEDC2, OTUD5, USP3, UBE3B,C10ORF46, USP9X, USP4, EDEM3, C12ORF51, STUB1, EDEM1, MYCBP2, UBE2R2,SUMO2, RNF123, SQSTM1, MAP3K1, USP39, PPP2CB, USP36, FBXW11, USP33,TRIP12, BUB3, USP31, UBXN1, STAMBP, LRRC41, CBL, SPSB2, BIRC6, UBE2Q2,UBE2Q1, CCNB1, WSB1, PSMD14, HSP90B1, GMCL1, PSMD13, CUL4A, TCEB2,TBL1X, and BARD1); GO:0051603, proteolysis involved in cellular proteincatabolic process (IDE, CBX4, MYLIP, CDC16, CD2AP, ZNRF2, WWP2, CUL9,RABGEF1, RBCK1, RNF149, RNF34, AUP1, CDCA3, PAN2, ANAPC1, USP1, RELA,UBR4, SKP2, SOCS7, UBR2, UBE2J2, PSMA2, MIB1, PSMA1, UHRF1, KDM2A,PIAS4, TRIM33, FBXL8, UBR5, PSMA4, ASB1, FBXL5, RNF138, FBXL4, PIAS2,TNFAIP3, FBXL3, ASB6, RAD23B, RAD23A, NEDD8, UBA5, ANAPC11, CDC34,PSMA7, UBAC1, PSMB5, ARIH1, PSMB4, ARIH2, UBE2D2, PSMB1, FBXW5, FBXW4,FBXO43, STAMBPL1, RNF168, TRAF7, RNF167, FBXO9, HECTD1, FBXO7, HACE1,PCNP, CDC20, CDC26, ATG3, ATE1, URM1, RNF6, PSMC5, PSMC4, VCP, UBA1,OTUB1, UBA2, UBA3, KIAA0368, UBE2G1, UBE2G2, BAP1, OS9, SENP6, FBXL19,FANCL, USP19, LONP1, MGRN1, PSMD1, PSMD2, PSMD3, USP11, NSMCE2, USP10,PSMD6, PSMD7, USP14, UBE2A, UFDIL, DDB1, HERC5, HERC4, UBE2I, ERLIN1,HERC3, HERC2, WDR48, SENP3, PSME1, UBE2K, PSME2, UBE2M, UBC, DDB2,USP21, ADAM17, KLHL12, SIAH2, USP22, USP24, UBE2S, USP7, CUEDC2, OTUD5,APH1A, USP3, UBE3B, C10ORF46, USP9X, USP4, EDEM3, C12ORF51, STUB1,EDEM1, MYCBP2, UBE2R2, SUMO2, RNF123, SQSTM1, MAP3K1, USP39, PPP2CB,USP36, FBXW11, USP33, TRIP12, BUB3, USP31, UBXN1, STAMBP, LRRC41, CBL,SPSB2, BIRC6, UBE2Q2, UBE2Q1, NCSTN, CCNB1, WSB1, PSMD14, HSP90B1,GMCL1, PSMD13, CUL4A, TCEB2, TBL1X, BARD1); and GO:0022403, cell cyclephase (ADCY3, PRC1, PKMYT1, RBM7, AURKB, CDC16, CD2AP, CDCA8, APP,RAD21, DDX11, INCENP, TARDBP, CDCA2, H2AFX, TUBG1, CCNA2, CDCA5, ASPM,CDCA3, ANAPC1, RAN, POLE, LIG3, SKP2, UBR2, ESPL1, TACC3, PPP1CB, WEE1,NCAPD3, NCAPD2, DCTN2, RCC2, TIMELESS, SPAG5, AKAP8, MAD2L2, DSCC1,MRE11A, KATNB1, ANAPC11, CDC34, RCC1, CCNG2, NIPBL, SAC3D1, NCAPG2,FBXO43, CLASP2, TCF3, HELLS, NUDC, SSSCA1, TAF1, MAP2K1, MKI67, PCNT,NUF2, CDC20, CDC26, RAD54L, ATM, HDAC3, CDKN1B, NOLC1, PLK1, ZNF318,SMC1A, ABL1, E2F1, MAD1L1, E2F4, RHOU, GTSE1, AKT1, CCNE1, SEH1L,PSMC3IP, RANBP1, DYNC1H1, FANCA, CDC7, ARHGEF2, CCNK, DSN1, CCNF, PAPD7,TPX2, NUSAP1, UBE2I, CDK6, RB1, RAD52, CDK4, CDK2, KLHDC3, RAD51, CCND1,CCND2, TOP3A, ADAM17, HAUS5, CAMK2G, USP9X, ZNF655, NCAPH, NCAPG, BCL2,NPAT, PAFAH1B1, GFI1, PPP3CA, BUB3, TRIP13, NFATC1, BOD1, TXNL4B, PDS5A,ILF3, MIS12, CDC25A, SMC4, CCNB1, PSMD13, CUL4A, GSPT1, SETD8, TXNL4A,and DNM2).

The candidate CRO for a particular gene can also be screened for “targetsite specificity,” where the chosen fragment of the extra-coding RNAhybridizes exclusively with the targeted genomic site (gene locus) butnot to any other genomic site (gene loci). Candidate transcripts can bescreened with any useful method. In one example, candidate sequences ofRNA oligonucleotides are assessed by using an algorithm with a genomicdatabase (e.g., using a BLAST algorithm with the following parameters:−E: 10, −B: 100, filter: dust, −W: 8, −M: 1, —N: −1, −Q: 2, and −R: 1).In another example, the candidate sequences are manually assessed byNorthern blot hybridization, as described in Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization with Nucleic Probes(ed. P. C. van der Vliet, Elsevier Science Publishers B.V., 1993).Optionally, the genomic database methods and Northern blot hybridizationmethods are performed under stringent conditions. Results from these twoexemplary approaches are shown in FIGS. 7A-7D and described herein.

In addition, the candidate CRO for a particular gene can be screened forits ability to bind DNMTs. For example, acceptable binding includes thatwhich results in an equal or higher affinity between the candidate CROand one or more DNMTs, as compared to the affinity between acorresponding DNA oligonucleotide and the one or more DNMTs. Affinitycan be assessed by any useful method. Exemplary methods include RNA/DNAelectrophoretic mobility shift assays (such as shown in FIGS. 4D-4F) andmeasurement of direct quantitative binding of radiolabeledoligonucleotides to DNMTs (e.g., as described in Burgisser, J. Recept.Res. 4:357-369, 1984). In particular embodiments, the CRO has bothtarget site specificity and the ability to bind DNMTs.

Different segments of an ecRNA can be tested to design the ssCRO,including those sequences upstream and downstream of the target gene andthose sequences corresponding to coding and intronic sections. When theecRNA includes a non-coding region, this region can be either upstreamor downstream of the coding region of a gene. For example, the ecRNA caninclude a sequence that is downstream of the polyA addition site of themRNA or in proximity to a promoter of the gene.

A CRO for a particular gene can be identified by any useful method. Forexample, the method includes one or more steps of identifying an ecRNAfor a particular gene, identifying a sequence having percentagecomplementarity to the ecRNA, and determining one or more binding sitesbetween the sequence having percentage complementarity and a DNMT (e.g.,DNMT1 or any other described herein), where the presence of bindingindicates that the sequence can reduce the activity of DNMT. Additionaloptional steps include determining the binding between the sequence ofthe CRO and the RNA or DNA for the gene; determining the methylationstatus of the target DNA, such as in one or more CpG islands; anddetermining the expression level of one or more genes. Furthermore, anyof these steps can be performed for more than one gene and/or in ahigh-throughput manner. These methods can be implemented by any usefultechnique, e.g., a RNA electrophoretic mobility shift assay (REMSA); invivo RNA immunoprecipitation (RIP) with ChIP-grade antibodies againstDNA methyltransferase; RNA immunoprecipitation with RNA deep sequencing(RIP-Seq); 5′, 3′-rapid amplification of the cDNA ends (RACE);microarray expression analysis (MEA); reduced representation bisulfitesequencing (RRBS); combined bisulfite restriction analysis (COBRA)assay; and combinations thereof.

Particular target genes can be identified in any useful manner. Forexample, ecRNAs corresponding to target genes can be down-regulated, andtheir effect on DNMT activity can be evaluated. Exemplary methods todown-regulate ecRNAs include using an RNAi agent, such as siRNA, dsRNA,miRNA, shRNA, or ptgsRNA. After confirming down-regulation of thetargeted RNAs, expression levels of the corresponding mRNAs can bemeasured by qRT-PCR, and methylation pattern of the corresponding geneloci can be characterized by bisulfite sequencing. Further studies caninclude in vivo transplantation assays.

In one example, the sequence of ecRNA can be determined by RNAimmunoprecipitation (RIP) followed by RNA deep sequencing (RIP-Seq). RIPis a technique used to detect the association of individual proteinswith specific RNA molecules in the cellular context. Generally, culturedcells are treated with formaldehyde to generate protein-RNA crosslinksbetween interacting molecules. Following immunoprecipitation of aprotein of interest and crosslink reversal, associated RNAs can berecovered, characterized, and quantitated by reverse transcriptasepolymerase chain reaction (RT-PCR). Then, the immunoprecipitated RNAswill be subjected to RNA-Seq, as described in Mortazavi et al., Nat.Methods 5:621-628, 2008, hereby incorporated by reference. RNA-Seq is arobust technology for monitoring expression by direct sequencing the RNAmolecules in a sample. Briefly, this methodology includes fragmentationof RNA to an average length of 200 nucleotides, conversion to cDNA byrandom priming, and synthesis of double-stranded cDNA (e.g., using theJust cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology).Then, the cDNA is converted into a molecular library for sequencing byaddition of sequence adapters for each library (e.g., fromIllumina®/Solexa), and the resulting 50-100 nucleotide reads are mappedonto the genome. RNA-Seq can be performed on RNAs immunoprecipitated byDNMT antibodies, using an IgG isotype antibody as a control. RIP-Seq canbe performed in multiple human and/or murine cell lines that express ordo not express a particular gene.

In another example, the methylation status of DNA can be determined byRBBS in a high-throughput manner, as described in Bock et al., Nat.Biotechnol. 28:1106-1114, 2010, incorporated herein by reference.Briefly, high-quality genomic DNA can be isolated from laboratory andprimary cell lines employed for RIP-Seq and digested with themethylation-insensitive enzyme MspI. Pre-annealed Illumina®/Solexasequence adapters containing 5′-methyl-cytosine can be ligated to theends of size-selected fragments. Adapter-ligated fragments can bebisulfite-converted, amplified by PCR, and then size-selected andsequenced on the Illumina/Solexa 1G Genome Analyzer. Finally, MEA can beperformed (e.g., using Affymetrix® GeneChip Human Gene 1.0 ST arrays) todetermine expression levels.

The efficacy and/or toxicity of CROs can be determined by any usefulmethod. Exemplary techniques include cellular assays performed in HL-60,U937, and/or K562 cell lines to determine one or more of changes inmethylation within the genomic region corresponding to the CRO, changesin methylation in unrelated sites as a control for an off-target effect,changes in expression level of the particular target gene, and changesin expression of unrelated genes as a control for an off-target effect.

Methods of Preparing a Chimeric RNA Oligonucleotide

The CRO can be prepared by any useful method. Exemplary methods includechemical synthesis (e.g., using a solid support), in vitrotranscription, digestion of long dsRNA by an RNase III family enzyme(e.g. Dicer, RNase III), and cell-based siRNA expression using plasmids,viral vectors, or PCR-derived expression cassette.

To incorporate modified nucleotides (e.g., cytidine analogs), thecorresponding nucleoside can be converted into building blocks, whichcan be incorporated by step-wise addition to a growing chain of theoligonucleotide sequence. Alternatively, di-, tri-, or tetra-nucleotideshaving one or more modified nucleotides can be converted into buildingblocks and incorporated into the oligonucleotide sequence. Exemplarybuilding blocks include cyanoethyl phosphoramidite or N-succinimideester building blocks. Examples of phosphoramidite building blocks ofmono-, di-, tri-, and tetra-nucleotides having one or more cytidineanalogs include5′-dimethoxytrityl(O-DMTr)-N4-dimethylformamidine-5,6-dihydro-5-aza-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (or5-aza-5,6-dihydro-dC-CE phosphoramidite);5″-O-DMTr-2′-deoxy-5-aza-cytidine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidite;and a dinucleotide including5′-O-DMTr-2′-deoxyguanosine-3′-O-cyanoethyl-N,N-diisopropylphosphoramiditeand5′-O-DMTr-2′-deoxy-5-aza-cytidine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidites.Additional cytidine analogs are described herein, and methods of makingcorresponding phosphoramidite building blocks are known.

The CROs can be used in the form of double-stranded chimeric RNAoligonucleotide (dsCRO). As described herein, the RNA transcript bindsDNMT1 and interferes with DNA methylation during transcription (FIG. 6A,left). In the absence of transcription, DNMT1 is not blocked and exertsenzymatic activity (FIG. 6A, right). For initial assays, dsCROs can betested to determine its ability to sequester DNMT1 (see FIG. 6B).Exemplary dsCROs include those having stem loop-like structures. Ascompared to 5-aza-2′-deoxycytidine, these dsCROs will likely be moreactive and less toxic. Though these dsCROs lack gene-specificity, theresults will provide preliminary binding data to DNMT. The followingtests can include developing corresponding single-stranded CROs todetermine gene-specific demethylating function.

Modifications to a RNA Oligonucleotide

The chimeric RNA oligonucleotide (CRO) can optionally includemodifications, such as, e.g., to increase binding to a DNMT, increasestability, or increase cellular uptake. In particular, the CRO caninclude one or more cytidine analogs to further enhance binding to DNMT.

Cytidine Analogs

The chimeric RNA oligonucleotide of the invention can optionally includeone or more modified cytidine nucleotides (or cytidine analogs). Asdescribed herein, cytidine analogs are useful in binding, and thusinactivating, DNMT.

Exemplary cytidine analogs includes 5-azacytidine and5-aza-2′-deoxycytidine and further analogs thereof, including thosehaving one of the following substitutions for the hydrogen of the4-amino group of the cytosine ring: methyl, ethyl, 9-fluorenylmethyl,9-(2-sulfo)fluorenylmethyl, 9-(2,7-dibromo)fluorenylmethyl,17-tetrabenzo[a,c,g,i]fluorenylmethyl, 2-chloro-3-indenylmethyl,benz[f]inden-3-ylmethyl,2,7-di-tert-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)methyl,1,1-dioxobenzo[b]thiophene-2-ylmethyl, 2,2,2-trichloroethyl,2-trimethylsilylethyl, 2-phenylethyl, 1-(1-adamantyl)-1-methylethyl,2-chloroethyl, 1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromethyl,1,1-dimethyl-2,2,2-trichlroethyl, 1-methyl-1-(4-biphenylyl)ethyl,1-(3,5-di-tert-butylphenyl)-1-methylethyl, 2-(2′- and 4′-pyridyl)ethyl,2,2-bis(4′-nitrophenyl)ethyl, N-(2-pivaloylamino)-1,1-dimethylethyl,2-[(2-nitrophenyl)dithio]-1-phenylethyl,2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, 2-adamantyl,vinyl, allyl, 1-isopropylallyl, cinnayl, 4-nitrocinnamyl,3-(3′-pyridyl)prop-2-enyl, 8-quinolyl, N-hydroxypiperidinyl,alkyldithio, benzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,p-chlorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl,9-anthrylmethyl, diphenylmethyl, 2-methylthioethyl,2-methylsulfonylethyl, 2-p-toluenesulfonyl)ethyl,[2-(1,3-dithianyl)]methyl, 4-methylthiphenyl, 2,4-dimethylthiphenyl,2-phosphonioethyl, 1-methyl-1-(triphenylphosphonio)ethyl,1,1-dimethyl-2-cyanoethyl, 2-dansylethyl, 4-phenylacetoxybenzyl,4-azidobenzyl, 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl,p-(dihydroxyboryl)benzyl, 5-benzisoxazolylmethyl,2-(trifluoroethyl)-6-chromonylmethyl, m-nitrophenyl,3,5-dimethoxybenzyl, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl,.alpha.-methylnitropiperonyl, o-nitrophenyl,3,4-dimethoxy-6-nitrobenzyl, phenyl(o-nitrophenyl)ethyl,2-(2-nitrophenyl)ethyl, 6-nitroveratryl, 4-methoxyphenacyl,3′,5′-dimethoxybenzoin, t-amyl, S-benzylthio, butynyl, p-cyanobenzyl,cyclohexyl, cyclopentyl, cyclopropylmethyl, p-decyloxybenzyl,diisopropylmethyl, 2,2-dimethoxycarbonylvinyl,o-(N,N-dimethylcarboxamido)henzyl,1,1-dimethyl-3-(N,N-dimethycarboxamido)propyl, 1,1-dimethylpropynyl,2-furanylmethyl, 2-iodoethyl, isobornyl, isobutyl, isonicotinyl,p-(p′-methoxyphenylazo)benzyl, 1-methylcyclobutyl, 1-methylcyclohexyl,1-methyl-1-cyclopropylmethyl, 1-methyl-1-(p-phenylazophenyl)ethyl,1-methyl-1-phenylethyl, 1-methyl-1-(4′-pyridyl)ethyl, phenyl,p-(phenylazo)benzyl, 2,4,6-tri-t-butylphenyl,4-(trimethylammonium)benzyl, 2,4,6-trimethylbenzyl, a urea (e.g., a ureawith phenothiazinyl-(10)-carbonyl, N′-p-toluenesulfonylaminocarbonyl,and N′-phenylaminothiocarbonyl), and an amide (e.g., formamide,acetamide, phenoxyacetamide, trichloroacetamide, trifluoroacetamide,phenyacetamide, 3-phenylpropamide, pent-4-enamide,o-nitrophenylacetamide, o-nitrophenoxyacetamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide,3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide,o-(benzoyloxymethyl)benzamide,2-[(t-butyldiphenylsiloxy)methyl)methyl]benzamide,3-(3′,6′-dioxo-2′,4′,5′-trimethylcyclohexa-1′,4′-diene)-3,3-dimethylpropionamide,o-hydroxy-trans-cinnamide, acetoacetamide, p-toluenesulfonamide, andbenzesulfonamide); or those having one of the following substitutionsfor the 4-amino group of the cytosine ring: 4-O-methoxy and4-S-methylsulfanyl. In some embodiments, the cytidine analog is furtherconjugated to a guanosine nucleotide (e.g., 2′-deoxyguanosine), such asfor 5-aza-2′-deoxycytidine-phosphodiester linkage-guanosine,5-aza-2′-deoxycytidine-phosphodiester linkage-2′-deoxy-guanosine,guanosine-phosphodiester linkage-5-aza-2′-deoxycytidine, and2′-deoxy-guanosine-phosphodiester linkage-5-aza-2′-deoxycytidine.Additional modifications to nucleotides are described herein.

Further Modifications to the Chimeric RNA Oligonucleotide

Modifications to the CRO include modified nucleotides having one or moremodifications to the chemical structure of the base, sugar, and/orbackbone, including the phosphodiester linker.

Non-limiting modified bases include those having 4- and/or 5-positionpyrimidine modifications, 8-position purine modifications, andmodifications at cytosine exocyclic amines. Additional modified basesrefer to nucleotide bases such as, for example, adenine, guanine,cytosine, thymine, uracil, xanthine, inosine, and queuosine that havebeen modified by the replacement or addition of one or more atoms orgroups. Further examples of modified bases include bases that arealkylated (e.g., as in O- and N-alkylated purines and pyrimidines),halogenated, thiolated, aminated, amidated, addition or removal of anaza group, or acetylated bases, individually or in combination.Exemplary modified bases include 5-propynyluridine, 5-propynylcytidine,6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine,2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine,5-methylcytidine, 5-methyluridine, 5-(2-amino)propyl uridine,5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine,7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine,5-methyloxyuridine, 7-deazaadenosine, 7-deazaxanthine, 7-deazaguanine,6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine,2-thiouridine, 4-thiouridine, 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, N6-methyladenosine,8-oxo-N6-methyladenine, 5-methylcarbonylmethyluridine, uridine5-oxyacetic acid, pyridine-4-one, pyridine-2-one, diaminopurine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine,5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,isoguanine, 8-substituted adenines and guanines, 5-substituted uracilsand thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,carboxyalkylaminoalkyl nucleotides, alkylcarbonylalkylated nucleotides,and universal bases (e.g., pyrrole, diazole, triazole, pyrene,pyridyloxazole, and pyrenylmethylglycerol derivatives, such as3-nitropyrrole or 5-nitroindole).

Exemplary modified sugars include 2-position sugar modifications, inwhich the 2-OH is replaced by a group such as an H, OR, R, halo (e.g.,F), SH, SR, NH₂, NHR, NR₂, or CN, wherein R is an alkyl moiety. Modifiedsugars also include, e.g., non-ribose sugars, such as mannose,arabinose, glucopyranose, galactopyranose, 4-thioribose, and othersugars, heterocycles, or carbocycles.

In some embodiments, the CRO includes a phosphorodiester linkage, suchas via 2′-ribose, as in the natural sugar phosphorodiester backbone inRNA. In other embodiments, to enhance the resistance to nucleasedegradation in vivo, the natural phosphorodiester linker—O—P(═O)(O)—O—CH₂— can be modified to be a phosphorothioate linker—O—P(═O)(S⁻)—O—CH₂—, a boranophosphate linker, or a methylphosphonatelinker. Phosphorothiate linkers can be implemented using standardphosphoramidite protocols and substituting a bis(O,O-diisopropoxyphosphinothioyl) disulfide (S-tetra) for iodine during the oxidationstep (see, e.g., Zon and Stec. “Phosphorothioate Analogues” inOligonucleotides and Their Analogs: A Practical Approach (ed. F.Eckstein, IRL Press, pp. 87-108, 1991); Zon, High Performance LiquidChromatography in Biotechnology (ed. W. S. Hancock, Wiley, New York, Ch.14, pp. 310-397, 1990); Stec et al., Tetrahed. Lett. 34:5317-5320, 1993;Iyer et al., J. Org. Chem. 55:4693-4699, 1990). Other exemplary linkersinclude phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, alkyl phosphonates (e.g., 3′-alkylenephosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g.,3′-amino phosphoramidate), aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates, andthionoalkylphosphotriesters. Other modifications to the backbone includethose replacing the phosphorous atom with short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages (e.g., morpholiniolinkages; siloxane backbones; sulfide, sulfoxide and sulphone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl andthioformacetyl backbones; alkene containing backbones; sulphamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts).

In other embodiments, the natural sugar phosphorodiester backbone can bereplaced with a protein nucleotide (PNA) backbone having repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. Other types ofmodifications for oligonucleotides designed to be more resistant tonuclease degradation are described U.S. Pat. Nos. 6,900,540 and6,900,301, incorporated herein by reference.

Therapeutic Agents

The CRO can include any useful therapeutic agent. Any of the therapeuticagents described below may be used in the compounds of the invention.Exemplary therapeutic agents for use in a CRO are demethylating agents(e.g. cytidine analogs, as described herein), DNA and/or RNA polymeraseinhibitors (e.g., a synthetic nucleoside that resembles cytidine, suchas cytarabine for the treatment of acute myelocytic leukemia;fludarabine for the treatment of hematologic malignancies by S-phasespecific inhibition of multiple DNA polymerases, including DNA primaseand DNA ligase I; gemcitabine; cladribine; and clofarabine); thymidylatesynthase inhibitors (e.g., 5-fluorouracil (5FU), floxuridine (FUDR),capecitabine, tegafur, and cannofur); immunosuppressants (e.g.,azathioprine); and other nucleoside analogs, such as thiopurines (e.g.,thioguanine and mercaptopurine) and adenosine analogs (e.g.,pentostatin, which is an adenosine deaminase inhibitor, and cladribine,which is a DNA polymerase inhibitor).

Demethylating Agents

The CRO can include one or more demethylating agents, includingnucleoside-based agent (e.g., a cytidine analog). Exemplarydemethylating agents include cytidine analogs, such as 5-azacytidine (orazacitidine), 5-aza-5,6-dihydrocytosine, 5-aza-2′-deoxycytidine (ordecitabine), beta-L-5-azacytidine, 2′-deoxy-beta-L-5-azacytidine,2′-deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (orN4-NPEOC-5-CdR), 5-fluorocytidine, 1-β-D-arabinofuranosil-5-azacytosine(or fazarabine), and 1-β-D-ribofuranosyl-2 (1H)-pyrimidinone (orzebularine); and non-nucleoside analogs, such as derivatives of4-aminobenzoic acid (e.g., procaine), epigallocatechin-3-gallate (EGCG),N-phthalyl-L-tryptophan (or RG108), and(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(curcumin).

Labels

A label can be linked to the chimeric RNA oligonucleotide to allow fordiagnostic and/or therapeutic treatment. Examples of labels includedetectable labels, such as an isotope, a radioimaging agent, a marker, atracer, a fluorescent label (e.g., rhodamine), and a reporter molecule(e.g., biotin).

Examples of radioimaging agents emitting radiation (detectableradio-labels) that may be suitable are exemplified by indium-111,technitium-99, or low dose iodine-131. Detectable labels, or markers,for use in the present invention may be a radiolabel, a fluorescentlabel, a nuclear magnetic resonance active label, a luminescent label, achromophore label, a positron emitting isotope for PET scanner, achemiluminescence label, or an enzymatic label. Fluorescent labelsinclude but are not limited to, green fluorescent protein (GFP),fluorescein, and rhodamine. Chemiluminescence labels include but are notlimited to, luciferase and β-galactosidase. Enzymatic labels include butare not limited to peroxidase and phosphatase. A histamine tag may alsobe a detectable label. For example, conjugates may comprise a carriermoiety and an antibody moiety (antibody or antibody fragment) and mayfurther comprise a label. The label may be for example a medicalisotope, such as for example and without limitation, technetium-99,iodine-123 and -131, thallium-201, gallium-67, fluorine-18, indium-111,etc.

Formation of a Complex with a Carrier

For delivery to the target gene, the chimeric RNA oligonucleotide of theinvention can covalently or non-covalently bind a carrier to form acomplex. The carrier can be used to alter biodistribution afterdelivery, to enhance uptake, to increase half-life or stability of theCRO (e.g., improve nuclease resistance), and/or to increase targeting toa particular cell or tissue type.

Exemplary carriers include a condensing agent (e.g., an agent capable ofattracting or binding a nucleic acid through ionic or electrostaticinteractions); a fusogenic agent (e.g., an agent capable of fusingand/or being transported through a cell membrane); a protein to target aparticular cell or tissue type (e.g., thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, or any other protein); a lipid; alipopolysaccharide; a lipid micelle or a liposome (e.g., formed fromphospholipids, such as phosphotidylcholine, fatty acids, glycolipids,ceramides, glycerides, cholesterols, or any combination thereof), ananoparticle (e.g., silica, lipid, carbohydrate, or otherpharmaceutically-acceptable polymer nanoparticle); a polyplex formedfrom cationic polymers and an anionic agent (e.g., a CRO), whereexemplary cationic polymers include polyamines (e.g., polylysine,polyarginine, polyamidoamine, and polyethylene imine); cholesterol; adendrimer (e.g., a polyamidoamine (PAMAM) dendrimer); a serum protein(e.g., human serum albumin (HSA) or low-density lipoprotein (LDL)); acarbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin,cyclodextrin, or hyaluronic acid); a lipid; a synthetic polymer, (e.g.,polylysine (PLL), polyethylenimine, poly-L-aspartic acid,poly-L-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolic) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymer,pseudopeptide-polyamine, peptidomimetic polyamine, or polyamine); acationic moiety (e.g., cationic lipid, cationic porphyrin, quaternarysalt of a polyamine, or alpha helical peptide); a multivalent sugar(e.g., multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, ormultivalent fucose); a vitamin (e.g., vitamin A, vitamin E, vitamin K,vitamin B, folic acid, vitamin B12, riboflavin, biotin, or pyridoxal); acofactor; or a drug to disrupt cellular cytoskeleton to increase uptake(e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin).

Diseases and Conditions

The CROs of the invention can be used to treat a variety of diseases andconditions involving aberrant DNMT activity. In particular, beneficialtreatments of cancer and imprinting disorders include use of the CROs toreduce DNMT activity in a gene-specific manner.

Cancer Therapy

The CROs of the invention may be used to treat any disease related toaberrant DNMT activity, such as cancer. In particular, the CROs of theinvention can be used to treat cancers having aberrant methylation ofspecific genes related with cancer progression. Exemplary cancersinclude those related to C/EBPa expression, such as myelodysplasticsyndrome (e.g., refractory anemia, refractory anemia with ringedsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation, refractory cytopenia withmultilineage dysplasia, myelodysplastic syndrome associated with anisolated del(5q) chromosome abnormality, and a myeloproliferativeneoplasm), leukemia (e.g., acute myeloid leukemia), head and neckcancer, liver cancer (e.g., hepatoma and hepatocellular carcinoma), lungcancer (e.g., adenocarcinoma and non-small cell lung cancer), prostatecancer (e.g., adenocarcinoma), and skin cancer (e.g., squamous cellcarcinoma); those related to SPI1, such as acute myeloid leukemia,T-cell lymphoma, and chronic lymphocytic leukemia-like disease; thoserelated to RXRA, such as non-small cell lung cancer (NSCLC); and thoserelated to RARB such as lung cancer (e.g., small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC)), head and neck cancer, breastcancer, prostate cancer, glioblastoma, and leukemia.

Other exemplary methylated genes and related cancers include RB1 inretinoblastoma (see Stirzaker et al., Cancer Res. 57:2229-2237, 1997);CDKN2A (INK 4A and ARF transcript) in lung and colon cancer (see Hermanet al., Cancer Res. 55:4525-4530, 1995; and Robertson et al., Mol CellBiol. 18:6457-6473, 1998); CDH1 in breast cancer, gastric cancer,thyroid cancer, leukemia, and liver cancer (see Graff et al., CancerRes. 55:5195-5199, 1995); CDH13 in lung cancer, ovarian cancer, andpancreatic cancer (see Toyooka et al., Cancer Res. 61:4556-4560, 2001);TIMP3 in brain cancer and kidney cancer (see Bachman et al., Cancer Res.59:798-802, 1999); VHL in kidney cancer (see Herman et al., Proc. NatlAcad. Sci. USA 91:9700-9704, 1994); MLH in colon cancer, endometrialcancer, and gastric cancer (see Kane et al., Cancer Res. 57:808-811,1997); MGMT in brain cancer, colon cancer, lung cancer, and breastcancer (see Qian et al., Cancer Res. 57:3672-3677, 1997); BRCA1 inbreast cancer and ovarian cancer (see Dobrovic et al., Cancer Res.57:3347-3350, 1997); GSTP1 in prostate cancer, liver cancer, coloncancer, breast cancer, and kidney cancer (see Lee et al., Proc. NatlAcad. Sci. USA 91:11733-11737, 1994); SMARCA3 in colon cancer andgastric cancer (see Moinova et al., Proc. Natl Acad. Sci. USA99:4562-4567, 2002); RASSF1 in lung cancer, liver cancer, and braincancer (see Dammann et al., Nat. Genet. 25:315-319, 2000); SOCS1 inliver cancer, colon cancer, and multiple myeloma (see Yoshikawa et al.,Nat. Genet. 28:29-35, 2001); ESR1 in colon cancer, breast estrogenreceptor-negative cancer, lung cancer, and leukemia (see Issa et al,Nat. Genet. 7:536-540, 1994), DAPK1 in lymphoma (see Katzenellenbogen etal., Blood 93:4347-4353, 1999). Additional cancers include thoseassociated with any gene described herein (e.g., a gene in cluster C).

Genetic Disorders

The CROs of the invention can be used to treat genetic disorders thatarise from aberrant DNA methylation, such as imprinting disordersrelated to uniparental disomy. The potency and the gene specificity ofthe CRO can be used to correct aberrant gain of DNA methylation within aspecific gene locus and/or to restore the normal expression of animprinted parental allele, as the expressed allele is mutated or deletedfor a particular locus.

Exemplary genetic disorders include imprinting disorders, such asBeckwith-Wiedemann Syndrome (BWS), Prader-Willi Syndrome (PWS), AngelmanSyndrome (AS), Albright hereditary osteodystrophy (AHO),pseudohypoparathyroidism type 1A (PHP-IA), and pseudohypoparathyroidismtype 1B (PHP-IB); disorders associated with loss of imprinting (LOI),which is considered the most abundant and most precocious alteration incancer, such as LOI in IGF2/H19 for Wilms' tumor; and repeat instabilitydiseases, where the expansion of trinucleotide (TNR) repeats leads tosilencing of the associated genes, such as in Fragile X syndrome andmyotonic dystrophy.

Administration and Dosage

The present invention also features pharmaceutical compositions thatcontain a therapeutically effective amount of a CRO of the invention.The composition can be formulated for use in a variety of drug deliverysystems. One or more physiologically acceptable excipients or carrierscan also be included in the composition for proper formulation. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences (Mack Publishing Company, Philadelphia, Pa.,17th ed., 1985). For a brief review of methods for drug delivery, see,e.g., Langer, Science 249:1527-1533, 1990.

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered parenterally (e.g., by intravenous,intramuscular, or subcutaneous injection), or by oral ingestion, or bytopical application or intraarticular injection at areas affected by thevascular or cancer condition. Additional routes of administrationinclude intravascular, intra-arterial, intratumor, intraperitoneal,intraventricular, intraepidural, as well as nasal, ophthalmic,intrascleral, intraorbital, rectal, topical, or aerosol inhalationadministration. Sustained release administration is also specificallyincluded in the invention, by such means as depot injections or erodibleimplants or components. Thus, the invention provides compositions forparenteral administration that include the above mention agentsdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a tablet, a capsule, andthe like. Furthermore, this invention provides compositions for localadministration, which may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount can be administered forprophylactic or therapeutic treatments. In prophylactic applications,compositions can be administered to a subject with a clinicallydetermined predisposition or increased susceptibility to cancer, or anydisease described herein. Compositions of the invention can beadministered to the subject (e.g., a human) in an amount sufficient todelay, reduce, or preferably prevent the onset of clinical disease. Intherapeutic applications, compositions are administered to a subject(e.g., a human) already suffering from disease (e.g., cancer, such asleukemia or a myelodysplastic syndrome) in an amount sufficient to cureor at least partially arrest the symptoms of the condition and itscomplications. An amount adequate to accomplish this purpose is definedas a “therapeutically effective amount,” an amount of a compoundsufficient to substantially improve some symptom associated with adisease or a medical condition. For example, in the treatment of acancer (e.g., those described herein), an agent or compound thatdecreases, prevents, delays, suppresses, or arrests any symptom of thedisease or condition would be therapeutically effective. Atherapeutically effective amount of an agent or compound is not requiredto cure a disease or condition but will provide a treatment for adisease or condition such that the onset of the disease or condition isdelayed, hindered, or prevented, or the disease or condition symptomsare ameliorated, or the term of the disease or condition is changed or,for example, is less severe or recovery is accelerated in an individual.

Amounts effective for this use may depend on the severity of the diseaseor condition and the weight and general state of the subject, butgenerally range from about 0.05 μg to about 1000 μg (e.g., 0.5-100 μg)of an equivalent amount of the agent per dose per subject. Suitableregimes for initial administration and booster administrations aretypified by an initial administration followed by repeated doses at oneor more hourly, daily, weekly, or monthly intervals by a subsequentadministration. The total effective amount of an agent present in thecompositions of the invention can be administered to a mammal as asingle dose, either as a bolus or by infusion over a relatively shortperiod of time, or can be administered using a fractionated treatmentprotocol, in which multiple doses are administered over a more prolongedperiod of time (e.g., a dose every 4-6 hours, 8-12 hours 14-16 hours,18-24 hours, every 2-4 days, every 1-2 weeks, and once a month).Alternatively, continuous intravenous infusions sufficient to maintaintherapeutically effective concentrations in the blood are contemplated.

The therapeutically effective amount of one or more agents presentwithin the compositions of the invention and used in the methods of thisinvention applied to mammals (e.g., humans) can be determined by theordinarily-skilled artisan with consideration of individual differencesin age, weight, and the condition of the mammal. Single or multipleadministrations of the compositions of the invention including aneffective amount can be carried out with dose levels and pattern beingselected by the treating physician. The dose and administration schedulecan be determined and adjusted based on the severity of the disease orcondition in the subject, which may be monitored throughout the courseof treatment according to the methods commonly practiced by cliniciansor those described herein.

The compounds of the present invention may be used in combination witheither conventional methods of treatment or therapy or may be usedseparately from conventional methods of treatment or therapy.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention in association with apharmaceutically acceptable excipient, as described herein, and anothertherapeutic or prophylactic agent known in the art.

In particular, combination therapies include a CRO and a histonedeacetylase (HDAC) inhibitor to further modulate transcription of genes,e.g., to promote the transcription of genes previously silenced byhypermethylation and acetylation of histones. Exemplary HDAC inhibitorsinclude hydroxamic acids (e.g., trichostatin A (ISA), vorinostat (SAHA),belinostat (PXD101),((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide(LAQ824), panobinostat (LBH589), subcroylanilidc hydroxamic acid (SAHA),oxamflatin, scriptaid, suberic bishydroxamic acid (SBHA),m-carboxy-cinnamic acid bishydroxamic acid (CBHA), or pyroxamide);cyclic peptides (e.g., trapoxin A, apidicin, TPX-HA, or depsipeptide(FR901228)); benzamides (e.g., entinostat (MS-275), N-acetyldinaline(CI994), or mocetinostat (MGCD0103); electrophilic ketones (e.g.,trifluoromethyl ketones or alpha-ketoamides, see Frey et al., Bioorg.Med. Chem. Lett. 12:3443-3447, 2002, and U.S. Pat. No. 6,511,990,incorporated herein by reference); and fatty acids (e.g., valproic acid,arginine butyrate, butyric acid, or phenylbutyrate).

EXAMPLES

Experimental Methods

The experimental methods described herein are used to obtain the resultsdiscussed in the brief description of the figures and the examplesdescribed herein, unless otherwise noted.

Cell culture: All cell lines were obtained from ATCC and grown inglutamine containing medium, at 37° C. in a humidified atmosphere with5% CO₂.

RNA isolation and Northern Blot Analysis: Total RNA isolation,electrophoresis, transfer, and hybridization were carried out asdescribed in Maniati et al., Molecular cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1982). Cytoplasmic RNA was isolatedwith the Paris kit (Ambion) according to the manufacturer'srecommendations. Nuclear RNAs were prepared according to Blobel, et al.,Science 154:1662-1665, 1966, with minor modifications. Briefly, equalamounts of viable cells (˜50 million) were washed with ice-cold PBSsupplemented with 5 mM vanadyl complex, 1 mM PMSF and resuspended in theice-cold lysis buffer: 1× Buffer A (10 mM HEPES-NaOH pH 7.6; 25 mM KCl;0.15 mM spermine; 0.5 mM spermidine; 1 mM EDTA; 2 mM Na butyrate); 1.25M sucrose; 10% glycerol; 5 mg/mL BSA; 0.5% NP-40; freshly supplementedwith protease inhibitors (2 mM leupeptin, add as ×400; 2 mM pepstatin,add as ×400; 100 mM benzamidine, add as ×400; a protease inhibitorcocktail (Roche Applied Science, Cat. No. 1836153), 1 tablet with 375 μLH₂O, add as ×100; 100 mM PMSF, add as ×100); 2 mM vanadyl complex (NewEngland Biolabs); and 20 units/mL RNase inhibitor (RNAguard; AmershamBiosciences). Samples were incubated at 0° C. for ˜10 minutes and passedthrough a Dounce homogenizer. The pelleted nuclei were resuspended in0.5 ml of lysis buffer and diluted with 2.25 mL of Dilution Buffer (2.13mL of “Cushion” buffer with 0.12 mL of 0.1 g/mL BSA), freshlysupplemented with protease inhibitors, overlaid onto 2 mL “cushions”(200 mL “Cushion” buffer consists of 15 mL ddH₂O; 15 mL 20× Buffer A; 30mL glycerol; 240 mL 2.5 M sucrose; freshly supplemented with proteaseinhibitors) into one SW 55 Ti tube, and centrifuged at 24,400 rpm (60minutes, 4° C.). The pelleted nuclei were resuspended in 1 mL Storagebuffer (1.75 mL ddH2O; 2 mL glycerol; 0.2 mL 20× Buffer A), freshlysupplemented with protease inhibitors. Nuclear RNAs were extracted asdescribed in Maniatis et al., 1982. All total, cytoplasmic, and nuclearRNA samples used in this study were treated with DNase I (10 U of DNaseI per 3 μg of total RNA; 37° C. for one hour; in the presence of RNaseinhibitor). After DNase I treatment, RNA samples were extracted withacidic phenol (pH 4.3) to eliminate any remaining traces of DNA.Polyadenylated and non-polyadenylated RNA fractions were selected withthe MicroPoly (A) Purist™ purification kit (Ambion). cDNA syntheses wereperformed with Random Primers (Invitrogen) with Transcriptor ReverseTranscriptase (Roche Applied Science) according to the manufacturer'srecommendation. cDNA was purified with a High Pure PCR ProductPurification Kit (Roche Applied Science). The sequences of variousNorthern Blotting probes were as follows:

C/EBPα mRNA: (SEQ ID NO: 10)5′-CCGCTCCTCCACGCCTGTCCTTAGAAAGGGGTGGAAACATAGGGACTTGGGGCTTGGAACCTAAGGTTGTTCCCCTAGTTCTACATGAAGGTGGAGGGTCTCTAGTTCCACGCCTCTCCCACCTCCCTCCGCACACACCCCACCCCAGCCTGCTATAGGCTGGGCTTCCCCTTGGGGCGGAACTCACTGCGATGGGGGTCACCAGGTGACCAGTGGGAGCCCCCACCCCGAGTCACACCAGAAAGCTAGGTCGTGGGTCAGCTCTGAGGATGTATACCCCTGGTGGGAGAGGGAGACCTAGAGATCTGGCTGTGGGGC-3′; and ecRNA: (SEQ ID NO: 11)5′-GTCACATTTGTAAATAATACAGCATTTTCCCTGGCGGCAATCCTGACTTTCATGAGCTCTCCATCCATCCTGAGCCCCTCTTACCCTAAGGGGGTGACTTACTTCCCCCAGGCAAGACAAATAAATAGCAGAGGACAAGGCTCCAAATGGAGTATGTCCAGAGCCTGAAGGCAGTCTCTTGGGGTCAGGGGAGGGGGCTGAAGGGGTTACTGGGCTGAGGCCTTGGCGAGGCTTCTTATCTGCCCCGGGGAGGAGGAGAGGGAGTCCTCTGCCTGAGGGGTAGGCCTGGCTAAGCAGCCCTAGGCTCAAGGAGCCCTTTGTGCAGACTTCCTTGCAAATCACCTACAGCTGCAGCCCTGGCCACTCACACACACCGCAGCTCCAGATICCAGCAGGACCCTCGGCCAGCAGGAAGAGGCCTCCAGTGGTAGGACCCTCCAACCCTCTCCTCTTTCCCTAGACCATGTGGCTACACCCTACC-3′.

qRT-PCR: Sybr green reactions were performed using iQ Sybr Greensupermix (Biorad, Hercules, Calif.) using the following parameters: 95°C. (10 min.), 40 cycles of 95° C. (15 sec.) and 60° C. (1 min.), and 72°C. (1 min.). TaqMan analysis was performed using Hotstart Probe One-stepqRT-PCR master mix (USB) at the following conditions: 50° C. (10 min.),95° C. (2 min.), and then 40 cycles of 95° C. (15 sec.) and 60° C. (60sec.). qRT PCR primers were located in the coding region for the mRNA(black double headed arrow) and after the polyA signal for the ecRNA(white double headed arrow) (FIG. 1A). The sequences of primers used forTaqMan real time PCR were as follows: human C/EBPa: Forward 5′-TCG GTGGACAAG AAC AG-3′ (SEQ ID NO:18), Reverse 5′-GCA GGC GGT CAT TG-3′ (SEQID NO:19), and Taqman Probe 5′-ACA AGGCCA AGC AGC GC-3′ (SEQ ID NO:20);ecRNA: Forward 5′-GGT TGT CTG TGG GCC AGG TCA-3′(SEQ ID NO:21), Reverse5′-AGA GCT CAT GAA AGT CAG GAT TG-3′ (SEQ ID NO:22), and Taqman Probe5′-AAT AAT ACAGCA TTT TCC CTG GCG G-3′(SEQ ID NO:23); human C/EBPγ:Forward: 5′-GGC TAG AGG AGC AGGTAC AT-3′ (SEQ ID NO:24), Reverse: 5′-GCCTGG GTA TGG ATA ACA CTA-3′ (SEQ ID NO:25), and Taqman Probe: 5′-CGACACCAC TCA TGT CAA TGG CTG-3′ (SEQ ID NO:26); human TP73: ABI Cat.#Hs01060631_m1; 18S rRNA: ABI Cat. #4310893E; and human 5S rRNA: ABICat. #Hs02385257_g1. The sequences of primers used for real-time RT PCR(Sybr) were as follows: human C/EBPa: Forward: 5′-CCG CTC CTCCAC GCC TGTCCT TAG-3′ (SEQ ID NO:27) and Reverse: 5′-GCC CCA CAG CCA GAT CTC TAGGTC-3′ (SEQ ID NO:28); ecRNA: Forward: 5′-TCA TGA GCT CTC CAT CCA TCCTGA-3′(SEQ ID NO:29) and Reverse: 5′-CTG GCCGAG GGT CCT GCT GGA ATC-3′(SEQ ID NO:30); and β-ACTIN Promega Cat. #G5740.

Primer extension and 5′,3′ RACE: cDNA from the HL-60 cell line wassynthesized as described above and run in alkaline condition (Sambrooket al., Molecular Cloning: A Laboratory Manual, 3rd ed., (Cold SpringHarbor Laboratory Press, 2001). Southern blot transfer and hybridizationwith oligo AL16 were performed as previously reported (Sambrook et al,2001). The sequences of primers used for 5′ RACE were as follows:R4—5′-AGA GGC GCG CTT GCC TAC AGG TGA-3′ (SEQ ID NO:12), R6—5′CTC GCCACT GGC GCT GAG GCC TGA-3′ (SEQ ID NO:13), and R8—5′-GAG TCT TGG GAG CCCTCAAGT GTC T-3′ (SEQ ID NO:14). The sequences of primers used for 3′RACE were as follows: AL21—5′-GTC ACA TTT GTA AAT AAT ACA GCA-3′ (SEQ IDNO:15), AL23—5′ CCC TGG CGG CAA TCC TGA CTT TCA-3′ (SEQ ID NO:16), andAL25—5′-TCA TGAGCT CTC CAT CCA TCC TGA-3′ (SEQ ID NO:17). 5′,3′ RACE wasperformed on two myeloid cell lines HL-60 and U937 using the ExactSTART™ Eukaryotic mRNA 5′- & 3′-RACE Kit according to the manufacturer'sinstructions.

Double thymidine block (early S-phase block): HL-60 cells were grownovernight to 70-80% confluence, washed twice with 1×PBS, and cultured inDMEM (10% FCS)+2.5 mM thymidine for 18 h (first block). Thymidine waswashed out with 1×PBS, and cells were grown in DMEM (10% FCS). After 8hours, cells were cultured in presence of thymidine for 18 h (secondblock) and then released as described. Synchrony was monitored by flowcytometry analysis of propidium iodide-stained cells using a LSRII flowcytometer (BD Biosciences) at the Harvard Stem Cell Institute/BethIsrael Deaconess Center flow cytometry facility.

DRB and ML-60218 treatment: After release from double thymidine block,HL-60 cells were treated with 100 μM of 5,6-Dichlorobenzimidazole1-β-D-ribofuranoside (DRB) (Sigma Aldrich) for 1, 2, and 3 hours. HL-60cells were treated with 25 μM ML-60218(2-chloro-N[5-(5-chloro-3-methyl-1-benzothiophen-2-yl)-2-methylpyrazol-3-yl]benzenesulfonamide,Calbiochem®) for 24 hours. Total RNA was harvested as described aboveand expression levels of C/EBPa, ecRNA, and 5S were measured by TaqmanqRT-PCR.

5′ Azacytidine (5 Aza-CR) treatment: K562 cells were cultured in thepresence of 10 μM 5Aza-CR (Sigma Aldrich) or 2% 1×PBS (mock treatment).Medium was refreshed every 48 hours. RNA (for RT-PCR) and genomic DNA(for bisulfite sequencing) were isolated after 7 days of treatment.

Down-regulation of ecRNA: Three different short hairpin RNAs targetingthe human ecRNA and one scrambled control were designed according to theDharmacon software program and cloned into the lentivirus vector pLKO.1(Sigma Aldrich), which has a puromycin selection marker. Lentivirusparticles were produced as previously described (Stewart et al., RNA9:493-501, 2003). HEK293T cells were co-transfected with either emptyvector or the pLKO-shRNA vector and Gag-Pol and Env constructs usingLipofectamine™2000 (Invitrogen) according to the manufacturer'srecommendation. Virus containing supernatants were collected 48 and 72hours after transfection and concentrated using a Centricon Plus-70100000 MWCO column (Millipore). Lentiviral transduction was performed inthe presence of Hexadimethrine bromide (final concentration 8 μg/ml) inthe human myeloid cell line U937. Puromycin (2 μg/ml) was added to thecultures two days after infection. Resistant clones were selected andscreened for down-regulation of the ecRNA by qRT-PCR. The short hairpinsRNAs sequences were as follows:

SC: (SEQ ID NO: 31) 5′-ATCTCGCTTGGGCGAGAGTAA-3′; Sh#1: (SEQ ID NO: 32)5′-AATAAATAGCAGAGGACAAGG-3′; Sh#2: (SEQ ID NO: 33)5′-CAGGCAAGACAAATAAATAGC-3′; and Sh#3: (SEQ ID NO: 34)5′-GAAGAGGCCTCCAGTGGTAGG-3′.

Up-regulation of ecRNA: The 3′ downstream region of C/EBPa ecRNA and anunrelated genomic region were cloned into the pBabe retrovirus vectorharboring a puromycin selection marker (Addgene plasmid 1764). K562cells were transfected with the Amaxa Cell Line Nucleofector® Kit V,Program T-003. Puromycin (2 μg/ml) was added to the cultures two daysafter transfection. Resistant clones were selected and screened forupregulation of ecRNA and the unrelated region (UR) by Northern BlotAnalysis. The primers for amplification of 3′ and 5′ regions (R1 and R2)and the Unrelated Region (UR) of ecRNA were as follows:

R1: (SEQ ID NO: 35) forward: 5′- ATG TCG GTGTCT TTT TAA AAC CAG -3′ and(SEQ ID NO: 36) reverse: 5′- GCT AAG CTT CCA GAG TGT AAA AGG -3′; R2:(SEQ ID NO: 37) forward: 5′- CCC GGC CCC AGA GTT AAG TTT GTC -3′ and(SEQ ID NO: 38) reverse: 5′- CGG CCC AGCTTT TAT ACC CGG CAG -3′; and UR:(SEQ ID NO: 39) forward: 5′- TA TAC AGC CAT GAA AGA AAC TTAC -3′ and(SEQ ID NO: 40) reverse: 5′- AGT TTT ACT GTG GTG TGT TTG TTC -3′.

Bisulfite Treatment, Combined Bisulfite Restriction Analysis Assay(COBRA) and Bisulfite Sequencing: The methylation profile of the C/EBPagene locus was performed by bisulfite sequencing as previouslydescribed. Briefly, 1 μg of genomic DNA was bisulfite converted by usingEZ DNA Methylation kit (Zymo Research, Orange, Calif.). The primers andPCR conditions for bisulfite sequencing and combined bisulfiterestriction analysis assay (COBRA) are summarized below. For COBRA, PCRproducts were purified and incubated with BstUI at 60° C. for 3 h. Thedigested DNA was then separated on a 3.5% agarose gel and stained withethidium bromide. For bisulfite sequencing, PCR products were gelpurified (Qiagen) and cloned into the pGEM®-T Easy Vector System(Promega). Sequencing results were analyzed using BiQ analyzer software.The sequences of primers for COBRA and bisulfite sequencing were asfollows: C/EBPa-1.4 kb region-: forward: 5′-GGT GTT TTT AGT TGT GTT TTTTT-3′ (SEQ ID NO:41) and reverse: 5′-AAA CCC TAA AAC CCC TTA-3′ (SEQ IDNO:42); C/EBPa Distal Promoter: forward: 5′-TAG TTT YGTTAG TTT GGG GGGTTT-3′ (SEQ ID NO:43) and reverse: 5′-TCT AAT CTC CAA ACT ACC CCT ATA-3′(SEQ ID NO:44); C/EBPa coding region: forward: 5′-AGG TTA AGG YGG TTGTGG GTT TTA-3′ (SEQ ID NO:45) and reverse:5′-CCA ACT ACT TAA CTT CAT CCTCCT-3′ (SEQ ID NO:46); C/EBPa 3′UTR: forward: 5′-AGG TTYGTG GTA GGA GGAGGG TTT A-3′ (SEQ ID NO:47) and reverse: 5′-TAA CCC ACR ACC TAA CTT TCTAAT-3′ (SEQ ID NO:48); TP73 promoter: forward: 5′-GTG GGY GGT TTY GTYGGG TTT TGT-3′(SEQ ID NO:49) and reverse: 5′-ACC CCT AAA CRA ATT ATA TAAA-3′ (SEQ ID NO:50); and C/EBPy promoter:

forward: (SEQ ID NO: 51) 5′- GAA GTG AATTTT TTA AAA TGA TTT -3′ andreverse: (SEQ ID NO: 52) 5′- TTT TGT TTT AGT TTT TTA AGT AGT TGG GA-3′.

MassARRAY: Quantitative DNA methylation analysis using the MassARRAYtechnique was performed by Sequenom, Inc., as previously described(Frommer et al., Proc Natl Acad Sci USA 89: 1827-1831, 1992). Briefly, 1μg of genomic DNA was converted with sodium bisulfite using the EZ DNAmethylation kit (Zymo Research, Orange, Calif.), FCR amplified, in vitrotranscribed, and then cleaved by RNase A. The samples were thenquantitatively tested for their DNA methylation status using MALDI-TOFmass spectrometry. The samples were desalted and spotted on a 384-padSpectroCHIP (Sequenom) using a MassARRAY nanodispenser (Samsung),followed by spectral acquisition on a MassARRAY Analyzer CompactMALDI-TOF MS (Sequenom). The resultant methylation calls were performedby the EpiTyper software v1.0 (Sequenom) to generate quantitativeresults for each CpG site or an aggregate of multiple CpG sites. Themethylation levels of aggregated multiple CpGs were calculated as themean of each CpGs methylation value and presented as percentage.

Nuclear RNA immunoprecipitation (nRIP): nRIP performed as described byEbralidze et al. (Science 303:383-387, 2004) with some modifications.Crosslinked nuclei were collected as follows: About 60×10⁶ HL-60 cellswere crosslinked with 1% formaldehyde (formaldehyde solution, freshlymade 50 mM HEPES-KOH; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 11%formaldehyde) for 10 minutes at room temperature. Crosslinking washalted by adding 1/10th volume of 2.66 M Glycine, kept for 5 minutes atroom temperature and 10 minutes on ice. Cell pellets were washed twicewith ice-cold PBS (freshly supplemented with 1 mM PMSF) and thenresuspended in cell lysis buffer (volume=4 mL, 1× Buffer of 10 mM TrispH 7.4; 10 mM NaCl; 0.5% NP-40, freshly supplemented with proteaseinhibitors (protease inhibitors cocktail: Roche Applied Science, Cat.No. 1836153, 1 tablet with 375 μL H₂O; add as ×100), 1 mM PMSF, and 2 mMvanadyl complex (NEB)). Cells were incubated at 0° C. for 10-15 minutesand homogenized by Dounce (10 strokes pestle A and 40 strokes pestle B).Nuclei were recovered by centrifugation at 2,000 rpm for 10 minutes at4° C. Nuclei were resuspended in 3 ml 1× Resuspension Buffer (50 mMHEPES-NaOH, pH 7.4; 10 mM MgCl₂) supplemented with 1 mM PMSF and 2 mMvanadyl complex. DNase treatment (250 U/ml) was performed for 30 minutesat 37° C., and EDTA (final concentration 20 mM) was added to halt thereaction. Resuspended nuclei were sonicated once for 20s (1 pulse every3 seconds) at 30% amplitude (Branson Digital Sonifer, Danbury, Conn.).

Immunoprecipitation: Before preclearing, the sample was adjusted to 1%Triton X-100; 0.1% sodium deoxycholate; 0.01% SDS; 140 mM NaCl; Proteaseinhibitors; 2 mM vanadyl complex; and 1 mM PMSF to facilitatesolubilization. In the preclearing step, ˜50 ul magnetic beads (ProteinA or G Magnetic Beads; #S1425S or #S143OS NEB) were added to the sampleand incubation was carried out for 1 h on a rocking platform at 4° C.Beads were removed in a magnetic field. The sample was divided intothree aliquots: (i) antibody of interest: DNMT1 antibody (Abcam cat#ab13537); (ii) preimmune serum: IgG (Sigma Aldrich); (iii) no antibody,no serum (input). About 5 μg antibody or preimmune serum was added tothe respective aliquot and incubation performed on a rocking platformovernight at 4° C. Input was stored at −20° C. after addition of SDS to2% final concentration. On Day II, About 200 μl of Protein A coatedsuper-paramagnetic beads (enough to bind 8 μg IgG) were added to thesamples and incubated on a rocking platform for 1 h at 4° C. Six washeswere performed with immunoprecipitation buffer (150 mM NaCl; 10 mMTris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA pH 8.0; 1% Triton X-100; 0.5%NP-40 freshly supplemented with 0.2 mM vanadyl complex and 0.2 mM PMSF)in a magnetic field. Proteinase K treatment to release DNA/RNA intosolution and to reverse HCHO crosslinking was performed in 200 μl of:100 mM Tris-HCl, pH 7.4; 0.5% SDS for the immunoprecipitated samples andin parallel for the input; Proteinase K, 500 μg/ml at 56° C. overnight.On Day III, beads were removed in magnetic field. Phenol (pH 4.3)extraction was performed after addition of NaCl (0.2 M finalconcentration). EtOH precipitation (in the presence of glycogen) wasconducted for 3 hours at −20° C. The pellet was dissolved in 180 μl H₂O,heated at 75° C. for 3 minutes, and immediately chilled on ice. Sampleswere treated with DNase I (250 U/ml) in the presence of RNase inhibitor300 U/ml in ×1 buffer #2 (NEB) at 37° C. for 30 minutes. Phenol (pH 4.3)extraction and EtOH precipitation were repeated. The RNA pellet wasdissolved in 50 μl H₂O.

Electrophoretic gel mobility shift assays (EMSAs) and Kd determination:DNA and RNA oligonucleotides (15 pmol) were end-labeled with [γ-32P] ATP(Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs).Reactions were incubated at 37° C. for 1h and then passed through G-25spin columns (GE Healthcare) according to the manufacturer'sinstructions to remove unincorporated radioactivity. Labeled sampleswere gel-purified on 10% polyacrylamide gels. Binding reactions werecarried out in 10 μL volumes in the following buffer: 5 mM Tris pH 7.4,5 mM MgCl₂, 1 mM DTT, 3% v/v glycerol, 100 mM NaCl. Various amounts ofpurified DNMT1 protein (BPS Bioscience Inc., 0.021-0.156 μM) wereincubated with 1.1 nM of 32P-labeled dsDNA and ss/ds RNAs. In thecompetitive assay, a fixed amount of protein and increasing amounts ofcompetitors (dsDNA or poly (dI-dC)) were used. All reactions wereassembled on ice and then incubated at room temperature. Samples wereloaded onto 6% native polyacrylamide gels (0.5×TBE) at 4° C. for 3 h at140 V. Gels were dried and exposed to X-ray film and a PhosphorImagerscreen. Data were analyzed with ImageQuant software. For affinityassays, the percent shifted species was determined as follows: themigration of the labeled DNA in this reaction was defined as zeropercent shifted and the ratio of the PhosphorImager counts in the areaof the lane above this band to the total counts in the lane was definedas background and subtracted from all other lanes. This band representedtotal input. Subsequent lanes containing DNMT1-nucleic acid complexeswere treated identically, and the percentage complex formation wascalculated as follows: [Input-(free probe for eachlane+background/input)]. All experiments contained a control reactionlacking DNMT1. The percentage complex formation was plotted as afunction of DNMT1 concentration using nonlinear regression analysisusing Prism 4.0a.

In vitro Transcription-Methylation Assay: The in vitrotranscription-methylation assays were performed on hemimethylated DNA(FIG. 14H) in the presence or absence of 5 U of human DNMT1 (New EnglandBiolabs) and 5 U of T7 RNA polymerase (Promega) or 5 U of E. coli RNApolymerase sigma-saturated holoenzyme (Epicentre). Reactions wereperformed in DNMT1 buffer according to the manufacturer'srecommendations supplemented with rNTPs and 1.25 mM MgCl₂, including the“DNMT1 only” reaction. This predetermined concentration of Mg²⁺ cationsis high enough to sustain activity of RNA polymerases and low enough notto inhibit DNMT1 activity. The primers for the in vitrotranscription/methylation assays were as follows: Forward: 5′-GGA AGGGCG ATC GGT GCG GGC CTC-3′ (SEQ ID NO:53), reverse (biotinylated):5′-Biotin-CAG CCC TCG AGG CCC GAA GCC ACC-3′ (SEQ ID NO:54), and reverse(not-biotinylated): 5′-CAG CCCTCG AGG CCC GAA GCC ACC-3′ (SEQ ID NO:55).

RNA immunoprecipitation sequencing (RIPseq): Total RNAimmunoprecipitated with DNMT1 antibody (Abcam cat #ab13537) and IgG(Sigma Aldrich) was processed for sequencing as described by Mortazaviet al. with some modifications. Double stranded cDNA was synthesizedusing the Just cDNA Double-Stranded cDNA Synthesis Kit (AgilentTechnology, Santa Clara, Calif.) according to the manufacturer'sinstructions. Illumina sequencing libraries were constructed from thesecDNA using a ChIP-Seq sample preparation kit (cat #IP-102-1001,Illumina, San Diego, Calif.) with minor modifications. Illuminapaired-end adaptor and PCR primers were used to replace the single readadaptor and primers in the kit. Constructed libraries were subjected toa final size-selection step on 10% Novex TBE gels (Invitrogen,Carlsbad). DNA fragments of 175-200 bp were excised from aSYBR-green-stained gel. DNA was recovered from the gel and quantifiedfollowing Illumina's qPCR quantification protocol. Paired end sequencingof these libraries was then performed on an Illumina GA IIx to achieve2×76 bp reads. Paired-End reads were trimmed to 50 bp and aligned to thereference genome hg19 using BWA59 with the following parameters: bwa aln−o 1 −l 25 −k 2; bwa sample −o 200. Then, the genome was divided intocourse bins (10 Kb) and reads were counted for DNMT1 RIP and IgG controlin each bin. A linear regression was fitted across all non-zero bins andthe slope of the regression was used as a scaling factor alpha tonormalize the RIP and control. Overlapping reads in the DNMT1 RIP wereaggregated into contiguous intervals. Each DNMT1 interval was tested forsignificance by comparing the number of reads within the interval thenumber of reads in the same region of the IgG control, multiplied by thescaling factor (exact binomial test, P=0.5). Multiple tests werecorrected by Benjamini-Hochberg. 16,187 intervals (representing thestart and end boundaries of peaks) were determined to be significantlyenriched in the DNMT1 RIP as compared to the IgG control (P<0.0001;q<0.0001). A false discovery rate of 7.5% was determined by determiningthe number of significantly enriched intervals in the IgGimmunoprecipitate using DNMT1 as a control. Significantly enriched DNMT1intervals have a mean length of 347 bp and a median of 67 reads perinterval. Every peak represents an interval with a ‘height’ value: thesum of all reads within an interval. All peaks were annotated withCEAS28 build on RefSeq hg19. A peak was considered belonging to a geneif located in the gene body or 3 kb up or downstream the gene (geneloci). Altogether, 6038 gene loci were covered by a least 1 significantRTPSeq peak.

Reduced representation bisulfite sequencing (RRBS): High quality genomicDNA was isolated from the myeloid cell line HL-60. DNA was digested withMsp1 (NEB), a methylation-insensitive enzyme that cuts C′CGG. DigestedDNA was size selected on a 4% NuSieve 3:1 Agarosegel (Lonza). For eachsample two slices containing DNA fragments of 40-120 bp and 120-220 bp,respectively, were excised from the unstained preparative portion of thegel. These two size fractions were kept apart throughout the procedureincluding the final sequencing. Pre-annealed Illumina adaptorscontaining 5′-methyl-cytosine instead of cytosine wereligated tosize-selected MspI fragments. Adapter-ligated fragments werebisulfite-treated using the EZ DNA Methylation kit (Zymo Research,Orange, Calif.). The products were PCR amplified, size selected, andsequenced on the Illumina GA IIx at a reading length of 36 bp.Sequencing reads were mapped to the reference genome hg19 usingRRBSmap61 allowing 2 mismatches. Reads from replicates were merged andprocessed using a custom computational pipeline. We considered only CpGlocated in regions with a depth of coverage greater than 3 reads. Theβ-score of CpG methylation in a given position is the ratio ofmethylated CpGs within the total number of CpGs through all reads.Levels of genes methylation is the mean of all CpG β-scores within −2 kbfrom the TSS to the end of first intron; for intronless genes the entiregene body was considered. Genes with less than 3 sequenced CpG in thepromoter or less than 3 sequenced CpG in the first exon-intron wereexcluded.

RNA expression profiling: RNA isolated from HL-60 cells was employed forsample amplification and labeling using the Whole Transcriptome assayreagent kits from Affymetrix. 10 μg of labeled RNA was hybridized onAffymetrix GeneChip Human Gene 1.0 ST array. Hybridization, washing,staining, and scanning were carried out as recommended by themanufacturer. Each hybridization reaction was performed in triplicates.Washes and staining were performed through the Fluidics Station 400 andthe GeneChip® Scanner 3000 (Affymetrix, Santa Clara, Calif., USA) wasused to measure the fluorescence intensity emitted by the labeledtarget. Raw data processing was performed using the Affymetrix GeneChip®Operating Software (GCOS). Microarrays were RMA normalized using ‘affy’,a R-Bioconductor library. C/EBPa expression was used as a threshold todefine expressing (>log₂ (4)) and not expressing (>log₂ (4)) genes forthe further analysis.

Data integration: We used the RefSeq transcripts database built on hg19(UCSC release) as a genome annotation reference for RipSeq, RRBS, andmicroarray expression experiments. We selected only the longesttranscripts. Accordingly, the number of 40857 RefSeq Ids was reduced to23250 transcript Ids. Then, we annotated all RIPSeq peaks against thegene loci which include exonic, intronic, and UTR regions plus 3 kbupstream of the TSS and 3 kb downstream of the Transcription End Site(TES) regions. We identified 6038 gene loci with DNMT1-RIPseq peaks and17212 gene loci without DNMT1-RIPseq peaks. Finally, we focused ourstudy on gene loci covered by the RRBS. We identified 4833 gene lociwith DNMT1-RIPseq peaks and covered by RRBS and 10973 gene loci withoutDNMT1-RIPseq peaks and covered by RRBS.

Gene ontology (GO): GO analysis was performed with DAVID. We focused ouranalysis on biological process annotations. GO enrichment was scoredusing the Benjamini corrected p-value.

Statistical analysis: Methylation changes of clones analyzed bybisulfate sequencing were calculated using the Fisher's exact test(GraphPad Prism Software Inc.). Methylation changes assessed byMassARRAY were calculated using paired t-test (GraphPad Prism SoftwareInc.). The statistical evaluation of DNMT1-RNA interaction versusexpression and methylation was estimated using the student t-test. Forboth populations: DNMT1-bound group and DNMT1-unbound group (box-plots;FIG. 15B), we measured the overrepresentation of genes which follow ourhypothesis (clusters B and C) against the ones which do not (clusters Aand D) using a 2-sample proportion test. P-values for t-test and2-sample proportion test were calculated by the R functions “t.tcst” and“prop.test” respectively. Values of P≤0.05 were considered statisticallysignificant. The mean±s.d. of two or more replicates is reported.

Data availability: Microarray expression, RIPseq and RRBS data areavailable on the gene omnibus database under the accession IDs GSE32153,GSE32162, and GSE32168 respectively.

The following examples are intended to illustrate, rather than limit theinvention.

Example 1 Characterization of Extra-Coding RNAs of C/EBPa

We hypothesized that non-coding RNAs (ncRNAs) may function to regulategene expression. To further test the universal character of thefunctionality of ncRNAs, we focused on the CCAAT enhancer bindingprotein alpha (C/EBPa) gene locus. C/EBPa is a master regulator in thehematopoietic system, and its expression is crucial during granulocyticdifferentiation. Impaired C/EBPa expression and/or function disruptsgranulopoiesis and contributes to leukemogenesis, and the presence ofC/EBPa mutations is now one of the criteria in the classification ofhuman Acute Myelogenous Leukemia (AML). In consideration of itsfundamental role in hematopoiesis, we decided to investigate theexistence of ncRNAs within the C/EBPa gene locus and to assess theirfunctional role in the gene expression.

As expected, we observed extensive “extra-coding” transcription arisingwithin the C/EBPa locus (FIGS. 1A-1C). Northern blot analysis of totalRNA from four leukemic cell lines, probing the region immediately afterthe C/EBPa polyadenylation site, revealed the presence of a major bandof ˜5 kb in HL-60 and U937, but not in K562 or Jurkat cell lines (FIGS.1A-1B). The identified transcript was distinct from the ˜2.6 kb signaldetected with a C/EBPa coding region probe, and correlated with C/EBPamRNA expression. These non-polyadenylated transcripts were enriched inthe nuclear fraction unlike polyadenylated C/EBPa mRNA (FIGS. 1C, 1H)suggesting that this RNA may have functional roles independent ofprotein coding potential. To map the entire length of these transcripts,we performed primer extension and 5′, 3′ RACE on total and polyA(−) RNAsisolated from HL-60 and U937 cells. That allowed us to identify thetranscriptional start site (TSS) of these long ecRNAs, one at −1.4 kband another at −0.8 kb upstream of the canonical C/EBPa mRNA TSS inHL-60 and U937 cell lines, respectively. Both novel transcriptsterminated at +3.6 kb downstream from the C/EBPa TSS (FIGS. 1I, 1J).These transcript(s) are “extra-coding” rather than non-coding, sincethey overlap the single C/EBPa exon and could potentially encode C/EBPaprotein. Theoretically, all regions of these extra-coding RNAs (ecRNAs)could potentially bind to DNMTs with high affinity. qRT-PCR analysisconfirmed concordant expression mode between extra-coding and codingtranscripts, in both cellular and nuclear RNAs (FIGS. 1D-1E).

To exclude the possibility that these long transcripts are unsplicedprecursors of the intronless C/EBPa gene, we examined the time course ofthe RNA synthesis and the RNA polymerase type(s) responsible for theinitiation of messenger and ecRNA. First, HL-60 cells synchronized bydouble thymidine block (thymidine arrests cells at the GO/G1 phaseboundary) were analyzed upon release from the block (with ˜85% of cellsentering the G1/S phase; FIG. 1K). Induction of ecRNA was observedimmediately after the block release, at marked contrast to significantlylower levels of C/EBPa mRNA at this time point (FIG. 1L). Thus, westipulated that C/EBPa ecRNA synthesis precedes the expression of itsoverlapping mRNA. Next, synchronized HL-60 cells were analyzed aftertreatment with the RNA polymerase II (Pol II) inhibitor DRB at differenttime points. In the presence of DRB, we observed strong down-regulationof mRNA but no decrease in ecRNA expression levels (FIG. 1F), suggestingthat the ecRNA transcription is mediated by a different RNA polymerasethan Pol II. To determine the effect of polymerase on ecRNA synthesis,we employed a specific RNA polymerase III (Pol III) inhibitor, ML-60218.Treatment with ML-60218 resulted in marked down-regulation of ecRNA,with little effect on C/EBPa mRNA (FIG. 1G). Collectively, thesefindings identify a novel Pol III-regulated RNA (i.e., ecRNA)overlapping the C/EBPa locus and preceding C/EBPa mRNA expression in Sphase.

Example 2 Functional Role of ecRNAs on mRNA Expression and DNAMethylation for C/EBPa

We also interrogated the functional role of ecRNAs for C/EBPa.Lentiviral shRNA-mediated down-regulation of ecRNAs led to significantdown-regulation of the mRNA (FIG. 2A). Furthermore, down-regulation ofecRNAs also affected the methylation status within the C/EBPa locus.When transcription of ecRNAs was reduced by shRNAs, DNA methylationlevels within the upstream promoter region were significantly increased(FIGS. 2B-2C). As shown in FIG. 2C, down-regulation of C/EBPa ecRNAsresulted in about 67%-92% increased methylation of CpG islands comparedto control with scrambled shRNA. C/EBPa expression can be highlysusceptible to methylation control, and hypermethylation in the CpGisland within the upstream promoter region of the C/EBPa gene has beenshown in a subclass of leukemia patients in the absence of other knowngenetic mutations. Based on these results, CROs can be used to targetecRNA and decrease methylation of tumor suppressor genes, such asC/EBPa.

In order to unveil the mechanistic aspects behind the control of DNAmethylation by transcription, we performed in vivo RNAimmunoprecipitation (RNA IP) with ChIP-grade antibodies against DNAmethyltransferase 1 (DNMT1). We repeatedly observed a significantenrichment of C/EBPa ecRNAs in the immunoprecipitated RNA fraction (FIG.3).

We also observed enrichment of other transcripts (e.g., extra-codingRNAs found in the PU.1 gene locus) and immunoprecipitation of other RNAswith DNMT1 antibody. Since RNA-DNMT1 binding appears not to besequence-specific, these complexes can be formed not only with C/EBPatranscripts but with other RNAs as well. Previous studies have shownthat downregulation of PU.1, by deletion of an upstream regulatoryelement (URE), induced acute myeloid leukemia, T-cell lymphoma, andchronic lymphocytic leukemia-like disease in a murine model (seeRosenbauer et al., Nat. Genet. 36:624-630, 2004; and Rosenbauer et al.,Nat. Genet. 38:27-37, 2006). Similarly, downregulation of PU.1 has beenobserved in myeloma cell lines and in a subset of freshly isolatedmyeloma cells. The 17-kb 5′ upstream enhancer (URE) and the promoterregion of the PU.1 gene were highly methylated in human myeloma celllines. Both sequences correspond to genomic regions giving rise todifferent ecRNAs (Ebralidze et al., Genes Dev. 22:2085-2092, 2008).Following 5-aza-2′-deoxycytidine treatment, upregulation of PU.1expression and correspondent growth arrest of myeloma cells have beenobserved (Tatetsu et al., Cancer Res. 67:5328-5336, 2007). Thedemethylating effect of CROs can be tested by its ability to restorePU.1 expression by correcting the aberrant methylation pattern in theURE and promoter region of myeloma cells. FIG. 9 shows exemplaryextra-coding RNAs (SEQ ID NOs:2-5) for use in generating CROs that cantarget SPI1 and reduce PU.1 methylation.

Example 3 Binding Interactions Between ecRNAs for C/EBPa and DNMT

To test the binding ability of DNMT1 to C/EBPa ecRNAs, we performed RNAelectrophoretic mobility shift assays (REMSA). We observed strongbinding of DNMT1 to folded RNAs comparable to that of double-strandedDNAs with the same primary sequence (FIGS. 4A-4E). Importantly,single-stranded RNA of the same primary sequence had almost undetectablebinding to DNMT1 (FIG. 4E). Thus, single-stranded CROs hound to itstarget sequence will bind DNMT, but unbound single-stranded CROs willnot bind (i.e., inactivate) DNMT. In this manner, gene-specificdemethylation is obtained.

Next, we compared REMSAs with fully folded and RNase T3 digested 250transcripts corresponding to CG-rich regions of the C/EBPa extra-codingRNA and the luciferase gene (FIG. 4F). A very similar pattern wasobtained for these two non-homologous sequences, strongly suggesting thenon-sequence-specific nature of the interaction between DNMT1 and RNA.Thus, the mechanism discovered for C/EBPa, as described herein, could beapplicable to other genes.

Finally, we tested the role of active transcription on DNMT1's abilityto methylate DNA. Using in vivo methylation assays, we determined themethylation status of hemi-methylated DNA in the presence or absence oftranscription. FIG. 5A(i)-(iv) shows various in vitro assays todetermine the effect of transcription on methylation status, where aparallel assay including polymerase and DNMT1 can be used to determinemethylation status during transcription (FIG. 5A(iii)) and another assayincluding DNMT1 can be used to determine methylation status in theabsence of transcription (FIG. 5A(iv)). Using combined bisulfiterestriction analysis (COBRA), methylated sites were converted intorestriction sites that can be digested by BstUI. FIG. 5B shows thatdigested products are absent when both DNMT1 and T7 polymerase arepresent, but digested products are present only when DNMT1 is present(black arrow). Thus, these data demonstrate that DNMT1 enzymaticactivity occurs in the absence of transcription and that RNAs can beused to prevent or reduce DNA methylation. Based on the experimentsdescribed herein, interactions between ecRNAs and other DNMTs, such asDNMT3a or DNMT3b, can also be tested.

Example 4 Screening for ecRNAs Having Target Site Specificity

To obtain transcripts having target site specificity, we performedgenomic database searches and Northern blot hybridization assays forecRNAs that hybridize exclusively with the targeted genomic site (genelocus) but not to any other genomic site (gene loci).

Using the BLAST genomic database, we searched for potentialhybridization sites for regions R1 and R2 within C/EBPa ecRNA (FIG. 1).R1 and R2 were assessed using the following BLAST parameters: −E: 10,−B: 100, filter: dust, −W: 8, −M: 1, −N: −1, −Q: 2, and −R: 1). Searchresults are provided for R2 (FIG. 7B) and R1 (FIG. 7C). Whereas R2hybridized to 1,732 candidates, R1 hybridized to only one. Accordingly,R1 but not R2 will likely bind with target site specificity.

Using Northern blot hybridization, we manually assessed whether R1and/or R2 would hybridize exclusively to one genomic site. As shown inFIG. 7D, probes corresponding to regions R1 and R2 were hybridized withtotal RNAs extracted from cells that express C/EBPa (i.e., HL-60 andU937 cell lines) and cells that lack C/EBPa (i.e., HEK293 and K562 celllines). The probe corresponding to R2 provided non-specifichybridization to all four cell lines. In contrast, the probecorresponding to R1 provided specific hybridization to C/EBPa-expressingcell lines HL-60 and U937.

Based on the results of these two methods, region R1 of C/EBPa would beappropriate for ssCRO design. Accordingly, these methods could be usedto determine other candidate transcripts having “target sitespecificity” for a targeted genomic site, such as one or more sites inSPI1 (spleen focus forming virus (SFFV) proviral integration oncogenespi1), RXRA (retinoid X receptor, alpha), RARB (retinoic acid receptor,beta), or any other gene described herein.

Example 5 Functional Studies of the ecRNA Regulating C/EBPa Expression

Further to Example 2 and to examine the functional role of the ecRNA inregulation of C/EBPa transcription on the C/EBPa locus, we performed RNAinterference-mediated loss-of-function and overexpression-mediatedgain-of-function experiments. Efficient knock-down of the ecRNA (˜4-folddecrease) achieved by small hairpin (sh) RNAs targeting the 3′ end ofthe ecRNA (but not including the C/EBPa mRNA) led to a decrease ofC/EBPa mRNA expression of similar magnitude (FIGS. 13A-13B), suggestingthat ecRNA may regulate C/EBPa expression. In view of the observationsthat increased methylation of C/EBPa gene promoter sequences has beenimplicated in leukemia and lung cancer, we set out to examine if therewas a connection between C/EBPa ecRNA and methylation of the C/EBPalocus. We analyzed methylation changes within the distal promoter(located at −0.8-0.6 kb from the C/EBPa transcription start site) bybisulfite sequencing (FIG. 13A). Intriguingly, ecRNA knockdown led to asignificant increase in DNA methylation levels, compared to thenon-targeting control (FIGS. 13C-13D).

To investigate whether enforced expression of the ecRNA was sufficientto inhibit methylation, the downstream region of the ecRNA (R1) and anunrelated region (UR; located 45 kb downstream of the gene) wereoverexpressed in K562 cells, which express ecRNA and C/EBPa mRNA at lowlevels (FIGS. 13L-13M). Ectopic expression of the downstream region ofecRNA resulted in significant increase in mRNA expression (FIG. 13E) andconcomitant decrease of DNA methylation in all three tested regionswithin the C/EBPa gene: the distal promoter, the C/EBPa coding region,and the C/EBPa 3′ UTR. This effect was specific to the ecRNA as we didnot observe changes in DNA methylation following overexpression of theunrelated region (UR) (FIG. 13F, 13N). Ectopic expression of theupstream region R2 of ecRNA did not affect C/EBPa expression, suggestinga modular character of the ecRNA (FIG. 13O). Indeed, analysis of the 3′part (R2) demonstrated its unique homology to the C/EBPa locus, whereasthe 5′ region (R1) shared sequence homology with thousands of genomicloci (FIGS. 7A-7D). Together, these loss- and gain-of-functionexperiments demonstrate that the presence of ecRNA prevents C/EBPa locusspecific DNA methylation.

To assess the specificity of the ecRNA overexpression, we tested whetherthe effect was confined to the C/EBPa locus. We compared expressionlevels with DNA methylation changes resulting from ecRNA overexpressionto that induced by the well-characterized hypomethylating agent5′-azacytidine (5-Aza-CR). We applied a high-throughput methylationanalysis of the C/EBPa locus using both a customized platform formassARRAY technology and direct bisulfite sequencing. Some 100 kbspanning both C/EBPa and the neighboring C/EBPg (C/EBP gamma, HGNC:1837,NCBI: NP_001797.1, NM_001806.2) gene were analyzed for both treatments(FIGS. 13G-13H). We did not observe major changes in methylation statusoutside the C/EBPa locus resulting from ecRNA overexpression (FIG. 13I).5-Aza-CR treatment led to the expected increase of C/EBPa mRNA levelsand reduced levels of DNA methylation of the C/EBPa locus (FIG. 13J). Incontrast, increased mRNA expression within the neighboring C/EBPg geneand changes in methylation status were achieved only after 5-Aza-CRtreatment (FIGS. 13J-13K) and not after R1 overexpression. In addition,to rule out off target effects on other chromosomes, we analyzed theexpression and methylation profile of the TP73 gene promoter, located onchr1p36, methylated and not expressed in K562, before and after the twotreatments. We detected expression and methylation changes exclusivelyafter 5-Aza-CR treatment (FIG. 13P). These results demonstrate that thisecRNA inhibits DNA methylation at the C/EBPa locus, with no discernibleeffects on DNA methylation at other tested loci.

Collectively, these results confirm the inverse link between ecRNA andC/EBPa methylation, and highlight the gene-specific, highly localizedeffect, supporting a specific cis regulatory role of the ecRNA on C/EBPaexpression. Accordingly, these experiments can be used to determine theeffect of ecRNAs on the methylation status of other candidatetranscripts, as well as to determine what regions of ecRNAs are mostappropriate for CRO design for selectively or specifically targeting anyof the genes described herein.

Example 6 Transcription Interferes with DNMT1 Methylation of DNA

To examine whether the act of transcription itself could interfere withthe ability of DNMT1 to methylate hemimethylated DNA, we performed acombined in vitro transcription and DNA methylation assay. Ahemimethylated DNA segment (bottom strand methylated) corresponding tothe 5′ end of the ecRNA (located at −1.4-0.5 kb from the C/EBPa TSS) wasengineered downstream of a T7 RNA polymerase promoter, and DNMT1methylase activity was monitored in the presence and absence of activetranscription (FIGS. 5A, 14C). In the absence of polymerase, there wasas expected a dramatic increase in DNA methylation of the upper strandmediated by DNMT1 (FIGS. 5A, 14A-14B). In contrast, no changes inmethylation were observed in the presence of both polymerase and DNMT1(FIGS. 5A-5B, 14B). These findings expand on previous studies that haveshown the ability of RNA to modulate DNMTs enzymatic activity in vitroand strongly suggest that RNAs arising from methylation-sensitive genesand their promoters can regulate corresponding gene expression byinterfering with DNA methylation.

Example 7 DNMT1 Binds to ecRNA with Greater Affinity than to DNA

We next sought to investigate the mechanism behind the observation thatthe C/EBPa methylation pattern is mediated by altered ecRNA levels.DNMT1 expression and enzymatic activity peaks during S phase.Intriguingly, increased ecRNA expression also occurs during the S phase.We therefore asked whether the presence of ecRNA during the S phase setthe stage for ecRNA interference with DNMT1 activity. To this aim, wetested the in vivo binding of DNMT1 to ecRNA by using RNAImmunoprecipitation (RIP) with an anti-DNMT1 antibody.

We observed ecRNA enrichment (>60-fold in HL-60 cells) in DNMT1-RNAprecipitates, as compared to the IgG control, demonstrating a physicalinteraction between ecRNA and DNMT1 (FIG. 15A). Similar results wereobtained in U937 cells (FIG. 15G). Analysis of polyA(+)/(−) fractions inDNMT1-RNA precipitates revealed enrichment of C/EBPa transcripts in thepolyA(−) fraction (FIG. 15H), suggesting that the major component ofC/EBPa transcripts in DNMT1-RNA precipitates was ecRNA. Next, we testedin vitro the ecRNA-DNMT1 interaction by performing RNA electrophoresismobility shift assays (REMSA) with regions corresponding to the 5′ and3′ end of the ecRNA (ssRNA: R05, corresponding to positions 4627-4648 ofSEQ ID NO:8, and R04, corresponding to positions 4777-4798 of SEQ IDNO:8) (FIGS. 15B, 15I).

First, to compare DNMT1 binding capacity of mismatched double-stranded(ds;R01/R03) and folded single-stranded (ss;R01, R04, and R05) RNAs todsDNAs (with the same primary sequence as the RNA oligonucleotides), weperformed competitive binding assay. We observed strong binding for bothds- and ssRNA in the presence of poly (dI-dC) (non-specific competitor)and dsDNA oligos (specific competitor) (FIGS. 15J, 15C).

Second, to investigate whether DNMT1 affinity for RNA was dependent onthe presence of CpG dinucleotides, we replaced the cytidine within CpGdinucleotides to uridine in the ssRNA oligonucleotide (R01: thesubstitution being neutral with regards to the predicted secondarystructures, according to RNAfold (FIG. 15K). The mutations did notdisrupt the RNA-protein complexes (FIG. 15L) unlike what has previouslybeen shown for DNA. Further, to test if the ecRNA-DNMT1 binary complexwas a case of trivial charge-charge interactions, we performed REMSA inthe presence of increasing concentrations of spermine, a molecule withfour positive charges at high density. Only a thousand-fold molar excessof spermine began to moderately affect the binding (FIG. 15M),suggesting a strong element of structural recognition indicate that theecRNA can physically associate with DNMT1. This interaction is notcontingent upon the presence of CpG dinucleotides, is not chargedependent, and requires certain RNA structural components.

Further to Example 3, we interrogated whether ecRNA displays higheraffinity for DNMT1 than DNA. We quantitatively compared the DNMT1binding affinity for ssRNA capable of forming secondary structures,unmethylated DNA (umDNA), hemimethylated DNA (hmDNA), and fullymethylated DNA (mDNA). RNA and DNA oligonucleotides at a constant molarconcentration were titrated with an increasing range of DNMT1 enzymeconcentrations using EMSA. An initial complex between enzyme and RNA wasformed with <0.013 μM DNMT1, whereas DNA began complexing at >0.026 μMDNMT1 (FIG. 15C). The dissociation constant (Kd) for RNA was 0.045(±0.004) μM, whereas for DNA was between 0.082 and 0.11 μM (umDNA 0.082(±0.03); hmDNA 0.14 (±0.11), and mDNA 0.11 (±0.06) μM)) (FIG. 15D).Remarkably, the DNMT1 complex with RNA was 2-fold stronger compared toumDNA and 3-fold stronger than hmDNA and mDNA. Importantly, RNA lackingstem-and-loop-like structures (R04; FIG. 15E) did not display the samebinding affinity as folded RNA (FIG. 15F), demonstrating the effect ofRNA secondary structure on RNA-DNMT1 complex formation.

In summary, these findings demonstrate that DNMT1 binds folded ecRNAwith higher affinity than DNA. Based on the experiments, regions of theecRNA in other candidate genes described herein, that can interact withDNMT1 and other DNMTs such as DNMT3a or DNMT3b, can be predicted basedon whether the particular region has a propensity to form a stable RNAsecondary structure. Further, knowing the effect of RNA secondarystructure on RNA-DNMT1 complex formation will aid in the design of CROsto target particular regions of the ecRNA, such as by testing thesecondary structure and binding stability of CRO-ecRNA complexes.

Example 8 Mapping the DNMT1 Epitranscriptome

Our observations described above suggested an inverse correlationbetween RNA-DNMT1 complexes and the methylation of the C/EBPa locus.Thus, we sought to explore whether this phenomenon mirrored a moreglobal mechanism of control of genomic methylation.

We applied a comparative genome-scale approach to identify and correlategene expression, association with DNMT1, and methylation status. cDNAlibraries made of RNAs that co-immunoprecipitated with anti-DNMT1antibody (DNMT1 library) and IgG (control library) were first assessedfor C/EBPa ecRNA enrichment (FIG. 16E) and subsequently analyzed bymassively parallel sequencing. Using 76-base paired-end sequencing, weproduced a total of 30.25 and 26.95 million pair reads for DNMT1 andcontrol libraries, respectively. All significant DNMT1 peaks wereannotated with CEAS build on RefSeq hg19 (a total of 16,176; P<0.0001;false discovery rate of 7.5%). These peaks were aligned against the hg19genome using locations scattered throughout the RefSeq genome annotationas follows: exonic 48%; intronic 23%; distal intergenic 5%; 5′ UTR 9%;3′ UTR 5%; and upstream promoter and downstream gene (assigned as 3 kbregions flanking the annotated genes) 10% (FIG. 16A).

We focused on the genomic regions encompassing 3 kb upstream anddownstream of the annotated genes (“gene loci”) and identified 6,042gene loci covered by the DNMT1 library. To assess the linkage betweengenomic loci giving rise to DNMT1-bound RNAs and the levels of genomicmethylation and expression of the corresponding nearby genes, we applieda genome-scale methylation screening (reduced representation bisulfitesequencing, RRBS) and gene expression microarray analysis. DNMT1 libraryand gene expression microarray database were aligned with the RRBSlibrary.

Within 15,794 RRBS-covered loci, 4,897 gene loci overlapped with theDNMT1-boudn library, and 10,897 gene loci did not (FIG. 16F). Withinboth groups, DNMT1-bound and not bound, genes were stratified accordingto expression and methylation levels (FIG. 16B). We observed that genesbelonging to the DNMT1-bound group had significantly higher levels ofexpression than genes belonging to the DNMT1 unbound group. An oppositetrend was observed for methylation levels: genes belonging to theDNMT1-bound group had significantly lower levels of methylation thangenes belonging to the DNMT1 unbound group. Thus, globally, DNMT1-RNAassociation is negatively correlated with gene locus methylation status.Next, we clustered genes within both groups according to levels ofexpression and methylation (FIG. 16C). We defined genes as “expressed”and “low or not expressed” above and below the score of log₂ (4)=2,respectively; and “hypomethylated” and “methylated” as genes with meanof all CpG scores below and above 50%, respectively. Interestingly, apredominant number of genes (i.e., more than 50%) were either in clusterB (i.e., DNMT1 unbound, methylation above 50%, and low expression levelbelow log₂ (4)=2) or in cluster C (i.e., DNMT1 bound, methylation below50%, and expression levels above or slightly below log₂ (4)=2). Forexample, 51.45% in the DNMT1 unbound group were in cluster B (lowerright quadrant of FIG. 16C) and 56.64% in the DNMT1 bound group clusterC (mid-lower and upper left quadrants of FIG. 16C), in agreement withthe proposed hypothesis that these interactions prevent DNMT1 dependentDNA methylation. An attractive feature of the epitranscriptome is itsdual applicability: (1) as a tool for basic and clinical oncology andstem cell research; and (2) as a guide for gene specific therapeuticapproaches.

Moreover, the numbers of genes in clusters B and C were significantlyhigher than numbers of genes in clusters A, F, E and G, H, D,respectively (P-value<0.0001). Examples of genes from clusters B and Care presented in FIGS. 16D, 16G, 16H. Grouping of genes in clusters A,F, E, G, H, and D may be the results of the technical limitation ofRRBS, contingent upon the genomic location of the restriction sites andthe DNA library size-selection, or these genes may be governed by yetanother mechanism of transcriptional control. Another interestingpossibility is that genes in clusters A, F, G, H, and D might be underlong-distance methylation control. It has recently been shown that thencRNA HOTTIP can exert regulatory function on distal genes viachromosomal looping, while being anchored to its own site oftranscription. Without wishing to be limited by mechanism, it ispossible that some of the DNMT1-bound RNAs identified by RIPseq willalign to sites of transcription and the respective local genes, and notto the gene loci where the actual DNMT1-binding event takes place (FIG.17). Interestingly, gene ontology analysis showed that genes inDNMT1-bound cluster C belong to multiple families of biologicalprocesses suggesting a general character of the DNMT1 sequestrationthroughout the genome (FIG. 18).

In conclusion, we have generated the first “DNMT1-centeredepitranscriptome”, a comprehensive map cross-referencingDNMT1-interacting transcripts to (i) DNA methylation and (ii) geneexpression. These data support the notion that RNA transcripts act as ashield sequestering DNMT1 and thus modulating DNA genomic methylation.The data also provide a wider scope for applications of CROs to target avariety of DNMT1-interacting RNA transcripts listed herein.

OTHER EMBODIMENTS

All publications, patent applications, and patents mentioned in thisspecification are herein incorporated by reference.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific desiredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the fields of medicine,pharmacology, or related fields are intended to be within the scope ofthe invention.

What is claimed is:
 1. A single-stranded synthesized RNA oligonucleotideconsisting of a nucleotide sequence corresponding to positions 4627-4648of SEQ ID NO:8, wherein said synthesized RNA oligonucleotide iscovalently or non-covalently linked to a targeting moiety or a carrier,wherein the carrier is a condensing agent, a fusogenic agent, a protein,a lipid, a lipopolysaccharide, a carbohydrate, a dendrimer, ananoparticle, a synthetic polymer, a vitamin, a cofactor, a cytoskeletaldisrupting drug, or a cationic moiety.
 2. A composition comprising thesynthesized RNA oligonucleotide of claim 1 and an excipient.
 3. Theoligonucleotide of claim 1, wherein said oligonucleotide furthercomprises a label selected from the group consisting of an isotope, aradioimaging agent or a radiolabel, a fluorescent label, a nuclearmagnetic resonance active label, a luminescent label, a chromophorelabel, a chemiluminescence label, an enzymatic label, a reportermolecule, an antibody, and an antibody fragment.
 4. The oligonucleotideof claim 1, wherein at least one nucleotide of said oligonucleotide is amodified nucleotide selected from the group consisting of 5-azacytidine,5-aza-2′-deoxycytidine, cytarabine, fludarabine, gemcitabine,clofarabine, azathioprine, 5-fluorouracil, floxuridine, mercaptopurine,thioguanine, pentostatin, and cladribine.
 5. The oligonucleotide ofclaim 1, wherein the targeting moiety is a cell-penetrating peptide. 6.The oligonucleotide of claim 1, wherein the carrier is said protein andthe protein targets a cell or tissue or is a serum protein.
 7. Theoligonucleotide of claim 1, wherein the condensing agent is a liposomeor a lipid micelle.
 8. The oligonucleotide of claim 1, wherein saidsynthesized RNA oligonucleotide binds to DNA methyltransferase 1(DNMT1).
 9. The oligonucleotide of claim 1, wherein the carrier is saidlipid and the lipid is a cholesterol.
 10. The oligonucleotide of claim1, wherein the carrier is said carbohydrate and the carbohydrate is apolysaccharide or a multivalent sugar.